PCG-1, a novel brown fat PPARgamma coactivator

ABSTRACT

The invention provides isolated nucleic acids molecules, designated PGC-1 nucleic acid molecules, which encode proteins which can modulate various adipocyte-associated activities including, for example, thermogenesis in adipocytes, e.g., brown adipocytes, and adipogenesis. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PGC-1 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a PGC-1 gene has been introduced or disrupted. The invention still further provides isolated PGC-1 proteins, fusion proteins, antigenic peptides and anti-PGC-1 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. application Ser. No.09/086,912 filed on May 29, 1998, and to U.S. provisional applicationNo. 60/048,107 filed on May 30, 1997, the contents of which areincorporated herein by reference.

GOVERNMENT SUPPORT

[0002] Work described herein was supported under grant 5R37DK31405awarded by the National Institutes of Health. The U.S. governmenttherefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Vertebrates possess two distinct types of adipose tissue: whiteadipose tissue (WAT) and brown adipose tissue (BAT). WAT stores andreleases fat according to the nutritional needs of the animal. BAT burnsfat, releasing the energy as heat (i.e., nonshivering heat). The uniquethermogenic properties of BAT reflect the activities of specializedmitochondria that contain the brown adipocyte-specific gene productuncoupling protein (UCP). Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419. UCP is a mitochondrial proton carrier that uncouplesrespiration from oxidative phosphorylation by collapsing the protongradient established from fatty acid oxidation without concomitant ATPsynthesis (Nicholls, D. and Locke, R. (1984) Physiol. Rev. 64:1-64).

[0004] UCP expression is tightly regulated, primarily by sympatheticnervous systems, in response to physiological signals, such as coldexposure and excess caloric intake (Girardier, L. and Seydoux, J. (1986)“Neural Control of Brown Adipose Tissue” In P. Trayhurn and D. Nichols(eds.) Brown Adipose Tissue (Arnold, London, 1986) pp. 122-151.Norepinephrine released from the local neurons interacts withβ-adrenergic receptors on the brown adipocyte cell membrane, causing anincrease in intracellular cyclic AMP (cAMP) levels (Sears, I. B. et al.(1996) Mol. Cell. Biol. 16(7):3410-3419). An increased level oftranscription of the UCP gene is a critical component in the cascade ofevents leading to elevated BAT thermogenesis in response to increasedcAMP (Kopecky, J. et al. (1990) J. Biol. Chem. 265:22204-22209;Rehnmark, S. M et al. (1990) J. Biol. Chem. 265:16464-16471; Ricquirer,D. F. et al. (1986) J. Biol. Chem. 261:13905-13910). BAT thermogenesisis used both (1) to maintain homeothermy by increasing thermogenesis inresponse to lower temperatures and (2) to maintain energy balance byincreasing energy expenditure in response to increases in caloric intake(Sears, I. B. et al. (1996) Mol. Cell. Biol. 16(7):3410-3419). Nearlyall experimental rodent models of obesity are accompanied by diminishedor defective BAT function, usually as the first symptom in theprogression of obesity (Himms-Hagen, J. (1989) Prog. Lipid Res.28:67-115; Himms-Hagen, J. (1990) FASEB J. 4:2890-2898). In addition,ablation of BAT in transgenic mice by targeted expression of a toxingene results in obesity (Lowell, B. et al. (1993) Nature 366:740-742).Thus, the growth and differentiation of brown adipocytes are keydeterminants in an animal's ability to maintain energy balance andprevent obesity (Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419).

[0005] Recently, several transcription factors have been identifiedwhich promote adipogenesis. These transcription factors includeCCAAT/enhancer binding protein (C/EBP) α, β, and δ and peroxisomeproliferator activated receptor (PPAR) γ. See Spiegelman, B. M. andFlier, J. S. (1996) Cell 87:377-389 for a review. C/EBP family memberssuch as C/EBPα, β, and δ play important roles in the regulation ofadipocyte-specific gene expression. For example, C/EBPα cantransactivate the promoters of several genes expressed in the matureadipocyte (Herrera, R. et al. (1989) Mol. Cell. Biol. 9:5331-5339;Miller, S. G. et al. (1996) PNAS 93:5507-551; Christy, R. J. et al.(1989) Genes Dev. 3:1323-1335; Umek, R. M. et al. (1991) Science251:288-291; Kaestner, K. H. et al. (1990) PNAS 87:251-255; Delabrousse,F. C. et al. (1996) PNAS 93:40964101; Hwang, C. S. et al. (1996) PNAS93:873-877). Overexpression of C/EBP α can induce adipocytedifferentiation in fibroblasts (Freytag, S. O. et al. (1994) Genes Dev.8:1654-1663) whereas expression of antisense C/EBPα inhibits terminaldifferentiation of preadipocytes (Lin, F. T and Lane, M. D. (1992) GenesDev. 6:533-544). The physiological importance of C/EBPα was furtherdemonstrated by the generation of transgenic, C/EBPα-knockout mice.Although adipocytes are still present in these animals, they accumulatemuch less lipid and exhibit decreased adipocyte-specific gene expression(Wang, N. et al. (1995) Science 269:1108-1112). C/EBPα was found to havea synergistic relationship with another transcription factor, PPARγ, inpromoting adipocyte differentiation (See Brun, R. P. et al. (1996) Curr.Opin. Cell Biol. 8:826-832 for a review). PPARγ is a nuclear hormonereceptor which exists in two isoforms (γ1 and γ2) formed by alternativesplicing (Zhu, Y. et al. (1995) PNAS 92:7921-7925) and which appears tofunction as both a direct regulator of many fat-specific genes and alsoas a “master” regulator that can trigger the entire program ofadipogenesis (Spiegelman, B. M. and Flier, J. S. (1996) Cell87:377-389). PPARγ forms a heterodimer with RXRα and has been shown tobind directly to well characterized fat-specific enhancers from theadipocyte P2 (aP2: Tontonoz, P. (1994) Genes Dev. 8:1224-1234) andphosphoenolpyruvate carboxykinase (PEPCK) genes (Tontonoz, P. (1994)Mol. Cell. Biol. 15:351-357).

[0006] Although the UCP gene promoter includes binding sites for C/EBP(Yubero, P. et al. (1994) Biochem. Biophys. Res. Commun. 198:653-659)and a PPARγ-responsive element (Sears, I. B. et al. (1996) Mol. Cell.Biol. 16(7):3410-3419), C/EBP and PPARγ do not seem to be sufficient toinduce UCP expression (Sears, I. B. et al. (1996) Mol. Cell. Biol.16(7):3410-3419). It would be highly desirable, therefore, to identify apossible additional factor which acts in combination with either C/EBPor PPARγ to activate UCP expression and thus to promote BATthermogenesis.

SUMMARY OF THE INVENTION

[0007] This invention is based, at least in part, on the discovery ofnucleic acid molecules which encode a family of novel molecules whichcan act in combination with PPARγ as a coactivator of UCP expression inBAT. These molecules are referred to herein as PPARγ Coactivator 1(“PGC-1”) proteins. Nucleic acid molecules encoding PGC-1 proteins arereferred to herein as PGC-1 nucleic acid molecules. The PGC-1 moleculesof the invention are capable of, for example, modulating adipogenesis,e.g., brown adipogenesis, and thermogenesis of a PGC-1 expressingtissue, e.g., BAT or muscle. Other functions of a PGC-1 family member ofthe invention are described throughout the present application.

[0008] Accordingly, one aspect of the invention pertains to isolatednucleic acid molecules (e.g., cDNAs) comprising a nucleotide sequenceencoding a PGC-1 protein or portions thereof (e.g., biologically activeor antigenic portions), as well as nucleic acid fragments suitable asprimers or hybridization probes for the detection of PGC-1-encodingnucleic acid (e.g., mRNA). In particularly preferred embodiments, theisolated nucleic acid molecule comprises the nucleotide sequence of SEQID NO:1, SEQ ID NO:4 or a nucleotide sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, or the codingregion or a complement of either of these nucleotide sequences.

[0009] In other particularly preferred embodiments, the isolated nucleicacid molecule of the invention comprises a nucleotide sequence whichhybridizes to or is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence shown inSEQ ID NO:1, SEQ ID NO:4 or a portion (e.g., 400, 450, 500, or morenucleotides) of this nucleotide sequence.

[0010] In yet another preferred embodiment, the nucleic acid moleculeincludes a nucleotide sequence encoding a protein having the amino acidsequence of SEQ ID NO: 2, SEQ ID NO:5. In yet another preferredembodiment, the nucleic acid molecule is at least 487 nucleotides inlength. In another preferred embodiment, the nucleic acid moleculecomprises a fragment of at least 487 nucleotides of the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:4 or a complement thereof. In afurther preferred embodiment, the nucleic acid molecule is at least 487nucleotides in length and encodes a protein having an PGC-1 activity (asdescribed herein).

[0011] Another embodiment of the invention features nucleic acidmolecules, preferably PGC-1 nucleic acid molecules, which specificallydetect PGC-1 nucleic acid molecules relative to nucleic acid moleculesencoding non-PGC-1 proteins. For example, in one embodiment, such anucleic acid molecule is at least 350, 400, 450, or 487 nucleotides inlength and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:4, or a complement thereof. In a particularly preferred embodiment,the nucleic acid molecule comprises a fragment of at least 487nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, or acomplement thereof. In preferred embodiments, the nucleic acid moleculesare at least 15 (e.g., contiguous) nucleotides in length and hybridizeunder stringent conditions to nucleotides 10214, 316, 515-532, 895-1279,1427-1456, 2325-2387 of SEQ ID NO:1. In other preferred embodiments, thenucleic acid molecules include nucleotides 1-28, 50-232, 518-535,895-1219, 2325-2386, 2975-3023 of SEQ ID NO:4.

[0012] In other preferred embodiments, the isolated nucleic acidmolecule encodes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 oran amino acid sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably 95% or more homologous to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:5. The preferred PGC-1 proteins of the present inventionalso preferably possess at least one of the PGC-1 biological activitiesdescribed herein.

[0013] In another embodiment, the isolated nucleic acid molecule encodesa protein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of SEQ ID NO:2, SEQ ID NO:5, e.g., sufficientlyhomologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 suchthat the protein or portion thereof maintains a PGC-1 activity.Preferably, the protein or portion thereof encoded by the nucleic acidmolecule maintains one or more of the following biologicalactivities: 1) it can interact with (e.g., bind to) PPARγ; 2) it canmodulate PPARγ activity; 3) it can modulate UCP expression; 4) it canmodulate thermogenesis in adipocytes, e.g., thermogenesis in brownadipocytes, or muscle; 5) it can modulate oxygen consumption inadipocytes or muscle; 6) it can modulate adipogenesis, e.g.,differentiation of white adipocytes into brown adipocytes; 7) it canmodulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα. In one embodiment, theprotein encoded by the nucleic acid molecule is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:5 (e.g., the entire aminoacid sequence of SEQ ID NO:2, SEQ ID NO:5).

[0014] In yet another embodiment, the isolated nucleic acid molecule isderived from a human and encodes a portion of a protein which includesone or more of the following domains or motifs: a tyrosinephosphorylation site, a cAMP phosphorylation site, a serine-arginine(SR) rich domain, an RNA binding motif, and an LXXLL (SEQ ID NO:3) motifwhich mediates interaction with a nuclear receptor. In another preferredembodiment, the isolated nucleic acid molecule is derived from a humanand encodes a protein (e.g., a PGC-1 fusion protein) which includes oneor more of the domains/motifs described herein and which has one or moreof the following biological activities: 1) it can interact with (e.g.,bind to) PPARγ; 2) it can modulate PPARγ activity; 3) it can modulateUCP expression; 4) it can modulate thermogenesis in adipocytes, e.g.,thermogenesis in brown adipocytes, or muscle; 5) it can modulate oxygenconsumption in adipocytes or muscle; 6) it can modulate adipogenesis,e.g., differentiation of white adipocytes into brown adipocytes; 7) itcan modulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα.

[0015] In another embodiment, the isolated nucleic acid molecule is atleast 15 nucleotides in length and hybridizes under stringent conditionsto a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:1, SEQ ID NO:4 or to a nucleotide sequence which is at least about50%, preferably at least about 60%, more preferably at least about 70%,yet more preferably at least about 80%, still more preferably at leastabout 90%, and most preferably at least about 95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4. Preferably,the isolated nucleic acid molecule corresponds to a naturally-occurringnucleic acid molecule. More preferably, the isolated nucleic acidencodes naturally-occurring human PGC-1 or a biologically active portionthereof. Moreover, given the disclosure herein of a PGC-1-encoding cDNAsequence (e.g., SEQ ID NO:1, SEQ ID NO:4), antisense nucleic acidmolecules (i.e., molecules which are complementary to the coding strandof the PGC-1 cDNA sequence) are also provided by the invention.

[0016] Another aspect of the invention pertains to vectors, e.g.,recombinant expression vectors, containing the nucleic acid molecules ofthe invention and host cells into which such vectors have beenintroduced. In one embodiment, such a host cell is used to produce PGC-1protein by culturing the host cell in a suitable medium. If desired, thePGC-1 protein can be then isolated from the medium or the host cell.

[0017] Yet another aspect of the invention pertains to transgenicnonhuman animals in which a PGC-1 gene has been introduced or altered.In one embodiment, the genome of the nonhuman animal has been altered byintroduction of a nucleic acid molecule of the invention encoding PGC-1as a transgene. In another embodiment, an endogenous PGC-1 gene withinthe genome of the nonhuman animal has been altered, e.g., functionallydisrupted, by homologous recombination.

[0018] Still another aspect of the invention pertains to an isolatedPGC-1 protein or a portion, e.g., a biologically active portion,thereof. In a preferred embodiment, the isolated PGC-1 protein orportion thereof can modulate thernogenesis in BAT. In another preferredembodiment, the isolated PGC-1 protein or portion thereof issufficiently homologous to an amino acid sequence of SEQ ID NO:2, SEQ IDNO:5 such that the protein or portion thereof maintains one or more ofthe following biological activities: 1) it can interact with (e.g., bindto) PPARγ; 2) it can modulate PPARγ activity; 3) it can modulate UCPexpression; 4) it can modulate thermogenesis in adipocytes, e.g.,thermogenesis in brown adipocytes, or muscle; 5) it can modulate oxygenconsumption in adipocytes or muscle; 6) it can modulate adipogenesis,e.g., differentiation of white adipocytes into brown adipocytes; 7) itcan modulate insulin sensitivity of cells, e.g., insulin sensitivity ofmuscle cells, liver cells, adipocytes; 8) it can interact with (e.g.,bind to) nuclear hormone receptors, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor; 9) it can modulatethe activity of nuclear hormone receptors; and 10) it can interact with(e.g., bind to) the transcription factor C/EBPα.

[0019] In one embodiment, the biologically active portion of the PGC-1protein includes a domain or motif, preferably a domain or motif whichhas a PGC-1 biological activity. The domain or motif can be a tyrosinephosphorylation site, a cAMP phosphorylation site, a serine-arginine(SR) rich domain, an RNA binding motif, and an LXXLL (SEQ ID NO:3) motifwhich mediates interaction with a nuclear receptor, or a combination ofone or more of these domains or motifs. Preferably, the biologicallyactive portion of the PGC-1 protein which includes one or more of thesedomains or motifs has one of the following biological activities: 1) itcan interact with (e.g., bind to) PPARγ; 2) it can modulate PPARγactivity; 3) it can modulate UCP expression; 4) it can modulatethermogenesis in adipocytes, e.g., thermogenesis in brown adipocytes, ormuscle; 5) it can modulate oxygen consumption in adipocytes or muscle;6) it can modulate adipogenesis, e.g., differentiation of whiteadipocytes into brown adipocytes; 7) it can modulate insulin sensitivityof cells, e.g., insulin sensitivity of muscle cells, liver cells,adipocytes; 8) it can interact with (e.g., bind to) nuclear hormonereceptors, e.g., the thyroid hormone receptor, the estrogen receptor,the retinoic acid receptor; 9) it can modulate the activity of nuclearhormone receptors; and 10) it can interact with (e.g., bind to) thetranscription factor C/EBPα.

[0020] The invention also provides an isolated preparation of a PGC-1protein. In preferred embodiments, the PGC-1 protein comprises the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:5 or an amino acid sequencewhich is at least about 50%, preferably at least about 60%, morepreferably at least about 70%, yet more preferably at least about 80%,still more preferably at least about 90%, and most preferably at leastabout 95% or more homologous to the amino acid sequence of SEQ ID NO:2,SEQ ID NO:5, e.g., the entire amino acid sequence of SEQ ID NO:2, SEQ IDNO:5. In other embodiments, the isolated PGC-1 protein comprises anamino acid sequence which is at least about 50%, preferably at leastabout 60%, more preferably at least about 70%, yet more preferably atleast about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5 and has one or more of the PGC-1biological activities described herein. Alternatively, the isolatedPGC-1 protein can comprise an amino acid sequence which is encoded by anucleotide sequence which hybridizes, e.g., hybridizes under stringentconditions, or is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence of SE Q IDNO:1, SEQ ID NO:4. It is also preferred that the preferred forms ofPGC-1 also have one or more of the PGC-1 biological activities describedherein.

[0021] The PGC-1 protein (or polypeptide) or a biologically activeportion thereof can be operatively linked to a non-PGC-1 polypeptide toform a fusion protein. In addition, the PGC-1 protein or a biologicallyactive portion thereof can be incorporated into a pharmaceuticalcomposition comprising the protein and a pharmaceutically acceptablecarrier.

[0022] The PGC-1 protein of the invention, or portions or fragmentsthereof, can be used to prepare anti-PGC-1 antibodies. Accordingly, theinvention also provides an antigenic peptide of PGC-1 which comprises atleast 8 amino acid residues of the amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:5 (or an amino acid sequence which is at least about 50%homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5) andencompasses an epitope of PGC-1 such that an antibody raised against thepeptide forms a specific immune complex with PGC-1. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues. The invention further provides an antibody that specificallybinds PGC-1. In one embodiment, the antibody is monoclonal. In anotherembodiment, the antibody is coupled to a detectable substance. In yetanother embodiment, the antibody is incorporated into a pharmaceuticalcomposition comprising the antibody and a pharmaceutically acceptablecarrier.

[0023] Another aspect of the invention pertains to methods formodulating a cell associated activity, e.g., proliferation,differentiation, survival, thermogenesis, oxygen consumption. Suchmethods include contacting the cell with an agent which modulates PGC-1protein activity or PGC-1 nucleic acid expression such that a cellassociated activity is altered relative to a cell associated activity(e.g., the same cell associated activity) of the cell in the absence ofthe agent. In a preferred embodiment, the cell associated activity isthermogenesis and the cell is a brown adipocyte. The agent whichmodulates PGC-1 activity can be an agent which stimulates PGC-1 proteinactivity or PGC-1 nucleic acid expression. Examples of agents whichstimulate PGC-1 protein activity or PGC-1 nucleic acid expressioninclude small molecules, active PGC-1 proteins, and nucleic acidsencoding PGC-1 that have been introduced into the cell. Examples ofagents which inhibit PGC-1 activity or expression include smallmolecules, antisense PGC-1 nucleic acid molecules, and antibodies thatspecifically bind to PGC-1. In a preferred embodiment, the cell ispresent within a subject and the agent is administered to the subject.

[0024] The present invention also pertains to methods for treatingsubjects having various disorders. For example, the invention pertainsto methods for treating a subject having a disorder characterized byaberrant PGC-1 protein activity or nucleic acid expression such as aweight disorder, e.g., obesity, anorexia, cachexia, or a disorderassociated with insufficient insulin activity, e.g., diabetes. Thesemethods include administering to the subject a PGC-1 modulator (e.g., asmall molecule) such that treatment of the subject occurs.

[0025] In one embodiment, the invention pertains to methods for treatinga subject having a weight disorder, e.g., obesity, or a disorderassociated with insufficient insulin activity, e.g., diabetes,comprising administering to the subject a PGC-1 activator, e.g., a PGC-1protein or portion thereof or a compound or an agent thereby increasingthe expression or activity of PGC-1 such that treatment of the diseaseoccurs. Weight disorders, e.g., obesity, and disorders associated withinsufficient insulin activity can also be treated according to theinvention by administering to the subject having the disorder a PGC-1activator, e.g., a nucleic acid encoding a PGC-1 protein or portionthereof such that treatment occurs.

[0026] The invention also pertains to methods for detecting geneticlesions in a PGC-1 gene, thereby determining if a subject with thelesioned gene is at risk for (or is predisposed to have) a disordercharacterized by abcrrant or abnormal PGC-1 nucleic acid expression orPGC-1 protein activity, e.g., a weight disorder or a disorder associatedwith insufficient insulin activity. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion characterized by an alterationaffecting the integrity of a gene encoding a PGC-1 protein, or themisexpression of the PGC-1 gene.

[0027] Another aspect of the invention pertains to methods for detectingthe presence of PGC-1 in a biological sample. In a preferred embodiment,the methods involve contacting a biological sample (e.g., acardiomyocyte, hepatocyte, neuronal cell, a brown adipocyte or a musclesample) with a compound or an agent capable of detecting PGC-1 proteinor PGC-1 mRNA such that the presence of PGC-1 is detected in thebiological sample. The compound or agent can be, for example, a labeledor labelable nucleic acid probe capable of hybridizing to PGC-1 mRNA ora labeled or labelable antibody capable of binding to PGC-1 protein. Theinvention further provides methods for diagnosis of a subject with, forexample, a weight disorder or a disorder associated with insufficientinsulin activity, based on detection of PGC-1 protein or mRNA. In oneembodiment, the method involves contacting a cell or tissue sample(e.g., a brown adipocyte sample) from the subject with an agent capableof detecting PGC-1 protein or mRNA, determining the amount of PGC-1protein or mRNA expressed in the cell or tissue sample, comparing theamount of PGC-1 protein or mRNA expressed in the cell or tissue sampleto a control sample and forming a diagnosis based on the amount of PGC-1protein or mRNA expressed in the cell or tissue sample as compared tothe control sample. Preferably, the cell sample is a brown adipocytesample. Kits for detecting PGC-1 in a biological sample are also withinthe scope of the invention.

[0028] Still another aspect of the invention pertains to methods, e.g.,screening assays, for identifying a compound for treating a disordercharacterized by aberrant PGC-1 nucleic acid expression or proteinactivity, e.g., a weight disorder or a disorder associated withinsufficient insulin activity. These methods typically include assayingthe ability of the compound or agent to modulate the expression of thePGC-1 gene or the activity of the PGC-1 protein thereby identifying acompound for treating a disorder characterized by aberrant PGC-1 nucleicacid expression or protein activity. In a preferred embodiment, themethod involves contact;ng a biological sample, e.g., a cell or tissuesample, e.g., a brown adipocyte sample, obtained from a subject havingthe disorder with the compound or agent, determining the amount of PGC-1protein expressed and/or measuring the activity of the PGC-1 protein inthe biological sample, comparing the amount of PGC-1 protein expressedin the biological sample and/or the measurable PGC-1 biological activityin the cell to that of a control sample. An alteration in the amount ofPGC-1 nucleic acid expression or PGC-1 protein activity in the ceilexposed to the compound or agent in comparison to the control isindicative of a modulation of PGC-1 nucleic acid expression and/or PGC-1protein activity.

[0029] The invention also pertains to methods for identifying a compoundor agent which interacts with (e.g., binds to) a PGC-1 protein. Thesemethods include the steps of contacting the PGC-1 protein with thecompound or agent under conditions which allow binding of the compoundto the PGC-1 protein to form a complex and detecting the formation of acomplex of the PGC-1 protein and the compound in which the ability ofthe compound to bind to the PGC-1 protein is indicated by the presenceof the compound in the complex.

[0030] The invention further pertains to methods for identifying acompound or agent which modulates, e.g., stimulates or inhibits, theinteraction of the PGC-1 protein with a target molecule, e.g., PPARγ,C/EBPα, a nuclear hormone receptor, e.g., the thyroid hormone receptor,the estrogen receptor, the retinoic acid receptor. In these methods, thePGC-1 protein is contacted, in the presence of the compound or agent,with the target molecule under conditions which allow binding of thetarget molecule to the PGC-1 protein to form a complex. An alteration,e.g., an increase or decrease, in complex formation between the PGC-1protein and the target molecule as compared to the amount of complexformed in the absence of the compound or agent is indicative of theability of the compound or agent to modulate the interaction of thePGC-1 protein with a target molecule.

BRIEF DESCRIPTION OF THE DRAWING

[0031] FIGS. 1A, 1A-1, and 1A-2 depict the mouse PGC-1 nucleotide (SEQID NO:1) and amino acid (SEQ ID NO:2) sequence.

[0032] FIGS. 2A-2B depict an analysis of the mouse PGC-1 sequence. Thefollowing domains are underlined in FIG. 2A: SR domains (amino acids565-598 and 617-631), an RNA-binding domain (amino acid 677-709), threeconsensus sites for phosphorylating protein kinase A (amino acids238-241, 373-376 and 655-668), and an LXXLL (SEQ ID NO:3) motif (aminoacids 142-146).

[0033]FIG. 2B is a schematic representation of the structure of mousePGC-1. Arrows indicate putative protein kinase A phosphorylation siteshaving the consensus sequence (R, K)2×(ST). The gray box indicates theSR rich region domain and black box indicates the RNA-binding domain.

[0034] FIGS. 3A-3B are bar graphs depicting the effect of mouse PGC-1 instimulating the transactivation of the UCP-1 promoter by PPARγ and thethyroid hormone receptor (TR). FIG. 3A depicts the increasedtranscription activation of the CAT reporter gene under the control ofthe UCP-1 promoter with respect to the indicated ligands/hormones inRAT1 IR cells. FIG. 3B is a graph depicting the increased transcriptionactivation of a reporter CAT gene under the control of UAS sequences(five copies) using mouse PGC-1 linked to GAL4 DBD.

[0035]FIG. 4 is a diagram of different mouse PGC-1 deletions to identifythe domain of PGC-1 which interacts with PPARγ. Indicated in the Figureare schematic representations of the PGC-1 deletions with thecorresponding percentage of input material that bound to PPARγ. TheLXXLL (SEQ ID NO:3) motif is located at amino acid residues 142-146. Theblack box corresponds to the PPARγ-binding domain of PGC-1 (amino acid292-338).

[0036]FIG. 5 is a diagram of different mouse PPARγ deletions to identifythe domain of PPARγ which interacts with PGC-1. Indicated in the Figureare schematic representations of the PPARγ deletions with thecorresponding percentage of input binding to PGC-1.

[0037]FIG. 6 is a bar graph depicting the effect in oxygen consumptionof chronic treatment of PGC-1 infected and control cells with cAMP andRetinoic Acid (RA).

[0038]FIG. 7 depicts the human PGC-1 nucleotide (SEQ ID NO:4) sequence.

[0039]FIG. 8 depicts the human PGC-1 amino acid (SEQ ID NO:5) sequence.

[0040]FIG. 9 depicts an alignment between the human PGC-1 amino acidsequence (SEQ ID NO:5) and the mouse PGC-1 amino acid sequence (SEQ IDNO:2). This alignment was performed with BLAST software found at theNational Center for Biotechnology Information (NCBI) web site (URL:http://www.ncbi.nlm.nih.gov, Altschul, S. F. et al. (1990) J Mol Biol215:403410; Madden, T. L. et al. (1996) Meth Enzymol 266:131-141) and itwas determined that human PGC-1 has a 94% identity to mouse PGC-1.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention is based on the discovery of novelmolecules, referred to herein as PGC-1 nucleic acid and proteinmolecules, which comprise a family of molecules having certain conservedstructural and functional features, and which play a role in or functionin adipocyte associated activities. The term “family” when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructual domain and having sufficient amino acid or nucleotide sequencehomology as defined herein. Such family members can be naturallyoccurring and can be from either the same or different species. Forexample, a family can contain a first protein of human origin, as wellas other, distinct proteins of human origin or alternatively, cancontain homologues of non-human origin. Members of a family may alsohave common functional characteristics.

[0042] In one embodiment, a PGC-1 molecule can modulate adipogenesis,e.g., adipogenesis of brown adipocytes and muscle cells. In anotherembodiment, a PGC-1 molecule can modulate thermogenesis in brownadipocytes. For example, a PGC-1 molecule of the invention can increasethermogenesis in adipocytes of an individual, thereby promoting weightloss in the individual. Thus, a PGC-1 molecule of the invention can beused to treat obesity. Additionally, the increase in thermogenicactivity caused by a PGC-1 molecule can also increase insulinsensitivity of the adipocytes as well as of muscle cells and livercells. Thus, a PGC-1 molecule of the invention can also be used to treatdisorders characterized by insufficient insulin activity such asdiabetes. Alternatively, inhibition of the activity of a PGC-1 moleculeof the invention can decrease thermogenesis in adipocytes of anindividual, thereby inhibiting weight loss in the individual. Thus, themodulators of PGC-1 molecules of the invention can be used to treatundesirable weight loss, e.g., cachexia, anorexia. Moreover, a PGC-1molecule of the invention can also be used as targets to screenmolecules, e.g., small molecules, which can modulate PGC-1 activity.PGC-1 molecule modulators can also be used to treat weight disorders,e.g., cachexia, anorexia, obesity, or disorders characterized byinsufficient insulin activity.

[0043] Mouse PGC-1 nucleic acid molecules were identified from mousebrown adipocytes based on their ability, as determined using a yeast twohybrid assay (described in Example I) to bind to PPARγ. As describedabove, PPARγ is a nuclear hormone receptor which functions as both adirect regulator of many fat-specific genes and also as a “master”regulator that can trigger the entire program of adipogenesis. Moreover,as the UCP gene promoter includes a PPARγ-responsive element, amodulator of PPARγ can modulate adipogenesis and UCP expression. UCPexpression can result in thermogenesis.

[0044] The nucleotide sequence of the mouse and human PGC-1 cDNA and thepredicted amino acid sequence of the mouse and human PGC-1 proteins areshown in FIGS. 1A, 1A-1, 1A-2, 2A, 7, and 8, and in SEQ ID NOs:1, 2, 4,and 5, respectively. Using all or a portion of the mouse nucleotidesequence (e.g., a 5′ portion of SEQ ID NO:1, e.g., nucleotides 1-50 ofSEQ ID NO:1) to probe a cDNA library from a human cell line such as ahuman muscle, heart, kidney, or brain cell line, the human PGC-1nucleotide sequence was obtained using routine experimentation asdescribed in Example II. The mouse PGC-1 gene, which is approximately3066 nucleotides in length, encodes a full length protein having amolecular weight of approximately 120 kD and which is approximately 797amino acid residues in length. The human PGC-1 gene, which isapproximately 3023 nucleotides in length, encodes a full length proteinhaving a molecular weight of approximately 120 kD and which isapproximately 798 amino acid residues in length. PGC-1 family memberproteins include several domains/motifs. These domains/motifs include:two putative tyrosine phosphorylation sites (amino acid residues 204-212and 378-385 of SEQ ID NO:2, and amino acid residues 205-213 and 379-386of SEQ ID NO:5), three putative cAMP phosphorylation sites (amino acidresidues 238-241, 373-376, and 655-658 of SEQ ID NO:2, and 239-242,374-377, and 656-658 of SEQ ID NO:5), a serine-arginine (SR) rich domain(anino acid residues 562-600 of SEQ ID NO:2, and 563-601 of SEQ IDNO:5), an RNA binding motif (amino acid residues 656-709 of SEQ ID NO:2,and 657-710 of SEQ ID NO:5), and an LXXLL motif (amino acids 142-146 ofSEQ ID NO:2, 143-147 of SEQ ID NO:5; SEQ ID NO:3) which mediatesinteraction with a nuclear receptor. As used herein, a tyrosinephosphorylation site is an amino acid sequence which includes at leastone tyrosine residue which can be phosphorylated by a tyrosine proteinkinase. Typically, a tyrosine phosphorylation site is characterized by alysine or an arginine about seven residues to the N-terminal side of thephosphorylated tyrosine. An acidic residue (asparagine or glutamine) isoften found at either three or four residues to the N-terminal side ofthe tyrosine (Patschinsky, T. et al. (1982) PNAS 79:973-977); Hunter, T.(1982) J. Biol. Chem. 257:48434848; Cooper, J. A. et al. (1984) J. Biol.Chem. 259:7835-7841). As used herein, a cAMP phosphorylation site is anamino acid sequence which includes a serine or threonine residue whichcan be phosphorylated by a cAMP-dependent protein kinase. Typically, thecAMP phosphorylation site is characterized by at least two consecutivebasic residues to the N-terminal side of the serine or threonine(Fremisco, J. R. et al. (1980) J. Biol. Chem. 255:42404245; Glass, D. B.and Smith, S. B. (1983) J. Biol. Chem. 258:14797-14803; Glass, D. B. etal. (1986) J. Biol. Chem. 261:2987-2993). As used herein, aserine-arginine rich domain is an amino acid sequence which is rich inserine and arginine residues. Typically, SR rich domains are domainswhich interact with the CTD domain of RNA polymerase II or are involvedin splicing functions. As used herein, an RNA binding motif is an aminoacid sequence which can bind an RNA molecule or a single stranded DNAmolecule. RNA binding motifs are described in Lodish, H., Darnell, J.,and Baltimore, D. Molecular Cell Biology, 3rd ed (W.H. Freeman andCompany, New York, N.Y., 1995). As used herein, an LXXLL (SEQ ID NO:3)refers to a motif wherein X can be any amino acid and which mediates aninteraction between an nuclear receptor and a coactivator (Heery et al.(1997) Nature 397:733-736; Torchia et al. (1997) Nature 387:677-684).

[0045] The PGC-1 protein is expressed in muscle, heart, kidney, brainand brown adipose tissue but not in white adipose tissue. In tissue fromcold acclimated animals, PGC-1 expression was highly induced in brownadipose tissue. Moreover, in tissue from cold acclimated animals, PGC-1expression was brown adipose tissue specific. PGC-1 expression intissues from cold acclimated animals parallels expression of UCP, thebrown adipose tissue marker responsible for the thermogenic activity ofthis tissue.

[0046] The PGC-1 protein or a biologically active portion or fragment ofthe invention can have one or more of the following activities: 1) itcan interact with (e.g., bind to) PPARγ; 2) it can modulate PPARγactivity; 3) it can modulate UCP expression; 4) it can modulatethermogenesis in adipocytes, e.g., thermogenesis in brown adipocytes, ormuscle; 5) it can modulate oxygen consumption in adipocytes or muscle;6) it can modulate adipogenesis, e.g., differentiation of whiteadipocytes into brown adipocytes; 7) it can modulate insulin sensitivityof cells, e.g., insulin sensitivity of muscle cells, liver cells,adipocytes; 8) it can interact with (e.g., bind to) nuclear hormonereceptors, e.g., the thyroid hormone receptor, the estrogen receptor,the retinoic acid receptor; 9) it can modulate the activity of nuclearhormone receptors; and 10) it can interact with (e.g., bind to) thetranscription factor C/EBPα.

[0047] Various aspects of the invention are described in further detailin the following subsections:

[0048] I. Isolated Nucleic Acid Molecules

[0049] One aspect of the invention pertains to isolated nucleic acidmolecules that encode PGC-1 or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify PGC-1-encoding nucleic acid (e.g., PGC-1 mRNA). Asused herein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolated PGC-1nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (e.g., a brown adipocyte). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

[0050] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4or a nucleotide sequence which is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, yet more preferablyat least about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4 or a portion thereof (e.g.,400, 450, 500, or more nucleotides), can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a human PGC-1 cDNA can be isolated from a humanheart, kidney, or brain cell line (from Stratagene, LaJolla, Calif., orClontech, Palo Alto, Calif.) using all or portion of SEQ ID NO:1, SEQ IDNO:4 as a hybridization probe and standard hybridization techniques(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or aportion of SEQ ID NO:1, SEQ ID NO:4 or a nucleotide sequence which is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, yet more preferably at least about 80%, still more preferablyat least about 90%, and most preferably at least about 95% or morehomologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4can be isolated by the polymerase chain reaction using oligonucleotideprimers designed based upon the sequence of SEQ ID NO:1, SEQ ID NO:4 orthe homologous nucleotide sequence. For example, mRNA can be isolatedfrom heart cells, kidney cells, brain cells, or brown adipocytes (e.g.,by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.(1979) Biochemistry 18: 5294-5299) and cDNA can be prepared usingreverse transcriptase (e.g., Moloney MLV reverse transcriptase,available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase,available from Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 or to thehomologous nucleotide sequence. A nucleic acid of the invention can beamplified using cDNA or, alternatively, genomic DNA, as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a PGC-1 nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

[0051] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:4 or a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4. The sequence ofSEQ ID NO:1 corresponds to the mouse PGC-1 cDNA This cDNA comprisessequences encoding the PGC-1 protein (i.e., “the coding region”, fromnucleotides 92 to 2482), as well as 5′ untranslated sequences(nucleotides 1 to 91) and 3′ untranslated sequences (nucleotides 2483 to3066). Alternatively, the nucleic acid molecule can comprise only thecoding region of SEQ ID NO:1 (e.g., nucleotides 92 to 2482) or thehomologous nucleotide sequence. The sequence of SEQ ID NO:4 correspondsto the human PGC-1 cDNA. This cDNA comprises sequences encoding thePGC-1 protein (i.e., “the coding region”, from nucleotides 89 to 2482),as well as 5′ untranslated sequences (nucleotides 1 to 88) and 3′untranslated sequences (nucleotides 2513 to 3023). Alternatively, thenucleic acid molecule can comprise only the coding region of SEQ ID NO:4(e.g., nucleotides 89 to 2482) or the homologous nucleotide sequence.

[0052] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleic acid molecule which is acomplement of the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4or a nucleotide sequence which is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, yet more preferablyat least about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4. A nucleic acid moleculewhich is complementary to the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:4 or to a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 is one which issufficiently complementary to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:4 or to the homologous sequence such that it canhybridize to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4or to the homologous sequence, thereby forming a stable duplex.

[0053] In still another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleotide sequence which is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, yet more preferably at least about 80%, still more preferablyat least about 90%, and most preferably at least about 95% or morehomologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4or a portion of this nucleotide sequence. In an additional preferredembodiment, an isolated nucleic acid molecule of the invention comprisesa nucleotide sequence which hybridizes, e.g., hybridizes under stringentconditions, to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4or to a nucleotide sequence which is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, yet more preferablyat least about 80%, still more preferably at least about 90%, and mostpreferably at least about 95% or more homologous to the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:4.

[0054] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the coding region of SEQ ID NO:1, SEQ ID NO:4 or thecoding region of a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, for example afragment which can be used as a probe or primer or a fragment encoding abiologically active portion of PGC-1. The nucleotide sequence determinedfrom the cloning of the PGC-1 gene from a mouse allows for thegeneration of probes and primers designed for use in identifying and/orcloning other PGC-1 family members, as well as PGC-1 homologues in othercell types, e.g. from other tissues, as well as PGC-1 homologues fromother mammals such as humans. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably at least about 25,more preferably about 40, 50 or 75 consecutive nucleotides of SEQ IDNO:1, SEQ ID NO:4 sense, an anti-sense sequence of SEQ ID NO:1, SEQ IDNO:4, or naturally occurring mutants thereof. Primers based on thenucleotide sequence in SEQ ID NO:1, SEQ ID NO:4 can be used in PCRreactions to clone PGC-1 homologues.

[0055] In an exemplary embodiment, a nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is about 100,preferably 100-200, preferably 200-300, more preferably 300400, and evenmore preferably 400487 nucleotides in length and hybridizes understringent hybridization conditions to a nucleic acid molecule of SEQ IDNO:1, SEQ ID NO:4.

[0056] Probes based on the PGC-1 nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a PGC-1 protein, such as by measuring a level ofa PGC-1-encoding nucleic acid in a sample of cells from a subject e.g.,detecting PGC-1 mRNA levels or determining whether a genomic P gene hasbeen mutated or deleted.

[0057] In one embodiment, the nucleic acid molecule of the inventionencodes a protein or portion thereof which includes an amino acidsequence which is sufficiently homologous to an amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5 such that the protein or portion thereofmaintains one or more of the following biological activities: 1) it caninteract with (e.g., bind to) PPARγ; 2) it can modulate PPARγ activity;3) it can modulate UCP expression; 4) it can modulate thernogenesis inadipocytes, e.g., thermogenesis in brown adipocytes, or muscle; 5) itcan modulate oxygen consumption in adipocytes or muscle; 6) it canmodulate adipogenesis, e.g., differentiation of white adipocytes intobrown adipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα.

[0058] As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO:2, SEQ ID NO:5) amino acid residues to an amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5 such that the protein or portion thereofmaintains one or more of the following biological activities: 1) it caninteract with (e.g., bind to) PPARγ; 2) it can modulate PPARγ activity;3) it can modulate UCP expression; 4) it can modulate thermogenesis inadipocytes, e.g., thermogenesis in brown adipocytes, or muscle; 5) itcan modulate oxygen consumption in adipocytes or muscle; 6) it canmodulate adipogenesis, e.g., differentiation of white adipocytes intobrown adipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα. In another embodiment, the protein is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to theentire amino acid sequence of SEQ ID NO:2, SEQ ID NO:5.

[0059] Portions of proteins encoded by the PGC-1 nucleic acid moleculeof the invention are preferably biologically active portions of thePGC-1 protein. As used herein, the term “biologically active portion ofPGC-1” is intended to include a portion, e.g., a domain/motif, of PGC-1that has one or more of the following activities: 1) it can interactwith (e.g., bind to) PPARγ; 2) it can modulate PPARγ activity; 3) it canmodulate UCP expression; 4) it can modulate the

enesis in adipocytes, e.g., thermogenesis in brown adipocytes, ormuscle; 5) it can modulate oxygen consumption in adipocytes or muscle;6) it can modulate adipogenesis, e.g., differentiation of whiteadipocytes into brown adipocytes; 7) it can modulate insulin sensitivityof cells, e.g., insulin sensitivity of muscle cells, liver cells,adipocytes; 8) it can interact with (e.g., bind to) nuclear hormonereceptors, e.g., the thyroid hormone receptor, the estrogen receptor,the retinoic acid receptor; 9) it can modulate the activity of nuclearhormone receptors; and 10) it can interact with (e.g., bind to) thetranscription factor C/EBPα.

[0060] Standard binding assays, e.g., immunoprecipitations and yeasttwo-hybrid assays as described herein, can be performed to determine theability of a PGC-1 protein or a biologically active portion thereof tointeract with (e.g., bind to) PPARγ, C/EBPα, and nuclear hormonereceptors. If a PGC-1 family member is found to interact with PPARγ,C/EBPα, and/or nuclear hormone receptors, then they are also likely tobe modulators of the activity of PPARγ, C/EBPα, and nuclear hormonereceptors.

[0061] To determine whether a PGC-1 family member of the presentinvention modulates UCP expression, in vitro transcriptional assays canbe performed. To perform such an assay, the full length promoter andenhancer of UCP can be linked to a reporter gene such as chloramphenicolacetyltransferase (CAT) and introduced into host cells. The same hostcells can then be transfected with PPARγ/RXRα and nucleic acid encodingthe PGC-1 molecule. The effect of the PGC-1 molecule can be measured bytesting CAT activity and comparing it to CAT activity in cells which donot contain nucleic acid encoding the PGC-1 molecule. An increase ordecrease in CAT activity indicates a modulation of UCP expression andsince UCP expression is known to be a critical component in the cascadeof events leading to elevated thermogenesis, this assay can also measurethe ability of the PGC-1 molecule to modulate thermogenesis inadipocytes.

[0062] The above described assay for testing the ability of a PGC-1molecule to modulate UCP expression can also be used to test the abilityof the PGC-1 molecule to modulate adipogenesis, e.g., differentiation ofwhite adipose tissue to brown adipose tissue, as UCP expression isspecific to brown adipose tissue. If a PGC-1 molecule can modulate UCPexpression is can most likely modulate the differentiation of whiteadipose tissue to brown adipose tissue. Alternatively, the ability of aPGC-1 molecule to modulate the differentiation of white adipose tissueto brown adipose tissue can be measured by introducing a PGC-1 moleculeinto a cell, e.g., a white adipocyte, and measuring the number ofmitochondria in the cell as compared to the number of mitochondria in acontrol cell which does not contain the PGC-1 molecule. As brownadipocytes are known to contain substantially greater numbers ofmitochondria than white adipocytes, an increase or decrease in thenumber of mitochondria (or in a mitochondrial marker such as cytochromec oxidase) in the test

as compared to the control cell indicates that the PGC-1 molecule canmodulate differentiation of white adipose tissue to brown adiposetissue.

[0063] The ability of a PGC-1 molecule to modulate insulin sensitivityof a cell can be determined by performing an assay in which cells, e.g.,muscle cells, liver cells, or adipocytes, are transformed to express thePGC-1 protein, incubated with radioactively labeled glucose (¹⁴Cglucose), and treated with insulin. An increase or decrease in glucosein the cells containing PGC-1 as compared to the control cells indicatesthat the PGC-1 can modulate insulin sensitivity of the cells.Alternatively, the cells containing PGC-1 can be incubated with aradioactively labeled phosphate source (e.g., [³²P]ATP) and treated withinsulin. Phosphorylation of proteins in the insulin pathway, e.g.,insulin receptor, can then be measured. An increase or decrease inphosphorylation of a protein in the insulin pathway in cells containingPGC-1 as compared to the control cells indicates that the PGC-1-canmodulate insulin sensitivity of the cells.

[0064] In one embodiment, the biologically active portion of PGC-1comprises a domain or motif. Examples of such domains/motifs include atyrosine phosphorylation site, a cAMP phosphorylation site, aserine-arginine (SR) rich domain, an RNA binding motif, and an LXXLL(SEQ ID NO:3) motif which mediates interaction with a nuclear receptor.In a preferred embodiment, the biologically active portion of theprotein which includes the domain or motif can modulate differentiationof white adipocytes to brown adipocytes and/or thermogenesis in brownadipocytes. These domains are described in detail herein. Additionalnucleic acid fragments encoding biologically active portions of PGC-1can be prepared by isolating a portion of SEQ ID NO:1, SEQ ID NO:4 or ahomologous nucleotide sequence, expressing the encoded portion of PGC-1protein or peptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of PGC-1 protein orpeptide.

[0065] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4(and portions thereof) due to degeneracy of the genetic code and thusencode-the same PGC-1 protein as that encoded by the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:4. In another embodiment, an isolatednucleic acid molecule of the invention has a nucleotide sequenceencoding a protein having an amino acid sequence shown in SEQ ID NO:2,SEQ ID NO:5 or a protein having an amino acid sequence which is at leastabout 50%, preferably at least about 60%, more preferably at least about70%, yet more preferably at least about 80%, still more preferably atleast about 90%, and most preferably at least about 95% or morehomologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5.

[0066] In addition to the mouse and human PGC-1 nucleotide sequencesshown in SEQ ID NO:1 and SEQ ID NO:4, it will be appreciated by thoseskilled in the art that DNA sequence polymorphisms that lead to changesin the amino acid sequences of PGC-1 may exist within a population(e.g., a mammalian population, e.g., a human population). Such geneticpolymorphism in the PGC-1 gene may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a PGC-1 protein, preferably a mammalian,e.g., human, PGC-1 protein. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of thePGC-1 gene. Any and all such nucleotide variations and resulting aminoacid polymorphisms in PGC-1 that are the result of natural allelicvariation and that do not alter the functional activity of PGC-1 areintended to be within the scope of the invention. Moreover, nucleic acidmolecules encoding PGC-1 proteins from other species, and thus whichhave a nucleotide sequence which differs from the mouse sequence of SEQID NO:1, SEQ ID NO:4, are intended to be within the scope of theinvention. Nucleic acid molecules corresponding to natural allelicvariants and human homologues of the mouse PGC-1 cDNA of the inventioncan be isolated based on their homology to the mouse PGC-1 nucleic aciddisclosed herein using the mouse cDNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions (see Example II).

[0067] Moreover, nucleic acid molecules encoding other PGC-1 familymembers and thus which have a nucleotide sequence which differs from thePGC-1 sequences of SEQ ID NO:1 or SEQ ID NO:4 are intended to be withinthe scope of the invention. For example, a PGC-2 cDNA can be identifiedbased on the nucleotide sequence of human PGC-1 or mouse PGC-1.Moreover, nucleic acid molecules encoding PGC-1 proteins from differentspecies, and thus which have a nucleotide sequence which differs fromthe PGC-1 sequences of SEQ ID NO:1 or SEQ ID NO:4 are intended to bewithin the scope of the invention. For example, rat PGC-1 cDNA can beidentified based on the nucleotide sequence of a human PGC-1.

[0068] Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention is at least 15 nucleotides in length andhybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or anucleotide sequence which is about 60%, preferably at least about 70%,more preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more homologous to thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4. In other embodiments,the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides inlength. As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1, SEQ ID NO:4 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein). In one embodiment, the nucleic acid encodesa natural human PGC-1.

[0069] In addition to naturally-occurring allelic variants of the PGC-1sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, thereby leading tochanges in the amino acid sequence of the encoded PGC-1 protein, withoutaltering the functional ability of the PGC-1 protein. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, SEQ ID NO:4. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of PGC-1 (e.g., thesequence of SEQ ID NO:2, SEQ ID NO:5) without altering the activity ofPGC-1, whereas an “essential” amino acid residue is required for PGC-1activity. For example, amino acid residues involved in the interactionof PGC-1 to PPARγ are most likely essential residues of PGC-1. Otheramino acid residues, however, (e.g., those that are not conserved oronly semi-conserved between mouse and human) may not be essential foractivity and thus are likely to be amenable to alteration withoutaltering PGC-1 activity.

[0070] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding PGC-1 proteins that contain changes in aminoacid residues that are not essential for PGC-1 activity. Such PGC-1proteins differ in amino acid sequence from SEQ ID NO:2, SEQ ID. NO:5yet retain at least one of the PGC-1 activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 60% homologous to the amino acid sequence of SEQID NO:2, SEQ ID NO:5 and is capable of modulating differentiation ofwhite adipocytes to brown adipocytes and/or thermogenesis of brownadipocytes. Preferably, the protein encoded by the nucleic acid moleculeis at least about 70% homologous, preferably at least about 80-85%homologous, still more preferably at least about 90%, and mostpreferably at least about 95% homologous to the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:5.

[0071] “Sequence identity or homology”, as used herein, refers to thesequence similarity between two polypeptide molecules or between twonucleic acid molecules. When a position in both of the two comparedsequences is occupied by the same base or amino acid monomer subunit,e.g., if a position in each of two DNA molecules is occupied by adenine,then the molecules are homologous or sequence identical at thatposition. The percent of homology or sequence identity between twosequences is a function of the number of matching or homologousidentical positions shared by the two sequences divided by the number ofpositions compared×100. For example, if 6 of 10, of the positions in twosequences are the same then the two sequences are 60% homologous or have60% sequence identity. By way of example, the DNA sequences ATTGCC andTATGGC share 50% homology or sequence identity. Generally, a comparisonis made when two sequences are aligned to give maximum homology. Unlessotherwise specified “loop out regions”, e.g., those arising from, fromdeletions or insertions in one of the sequences are counted asmismatches.

[0072] The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithim. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment paramethers include GAP Penalty=10, GapLength Penalty=10. For DNA alignments, the pairwise alignmentparamenters can be Htuple=2, Gap penalty-5, Window4, and Diagonalsaved=4. For protein alignments, the pairwise alignment parameters canbe Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5.

[0073] In a preferred embodiment, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch (J. Mol.Biol. (48):444-453 (1970)) algorithm which has been incorporated intothe GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1989) which has been incorporated into theALIGN program (version 2.0) (available athttp://vega.igh.cnrs.fr/bin/align-guess.cgi), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

[0074] An isolated nucleic acid molecule encoding a PGC-1 proteinhomologous to the protein of SEQ ID NO:2, SEQ ID NO:5 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or a homologousnucleotide sequence such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into SEQ ID NO:1, SEQ ID NO:4 or thehomologous nucleotide sequence by saundard teclmiques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in PGC-1 is preferably replaced with another aminoacid residue from the same side chain family. Alternatively, in anotherembodiment, mutations can be introduced randomly along all or part of aPGC-1 coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for a PGC-1 activity described hereinto identify mutants that retain PGC-1 activity. Following mutagenesis ofSEQ ID NO:1, SEQ ID NO:4, the encoded protein can be expressedrecombinantly (e.g., as described in Example IV) and the activity of theprotein can be determined using, for example, assays described herein.

[0075] In addition to the nucleic acid molecules encoding PGC-1 proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PGC-1 coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding PGC-1.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the entire coding region of SEQ ID NO:1 comprises nucleotides 92 to2482, the entire coding region of SEQ ID NO:4 comprises nucleotides 89to 2482). In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding PGC-1. The term “noncoding region” refers to 5′ and 3′sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0076] Given the coding strand sequences encoding PGC-1 disclosed herein(e.g., SEQ ID NO:1, SEQ ID NO:4), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of PGC-1 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of PGC-1 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of PGC-1 mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0077] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aPGC-1 protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An exampe of a route of administration of an antisensenucleic acid molecule of the invention includes direct injection at atissue site. Alternatively, an antisense nucleic acid molecule can bemodified to target selected cells and then administered systemically.For example, for systemic administration, an antisense molecule can bemodified such that it specifically binds to a receptor or an antigenexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecule to a peptide or an antibody which binds to a cellsurface receptor or antigen. The antisense nucleic acid molecule canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0078] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0079] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave PGC-1 mRNA transcripts to thereby inhibittranslation of PGC-1 mRNA. A ribozyme having specificity for aPGC-1-encoding nucleic acid can be designed based upon the nucleotidesequence of a PGC-1 cDNA disclosed herein (e.g., SEQ ID NO:1, SEQ IDNO:4). For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in aPGC-1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 andCech et al. U.S. Pat. No. 5,116,742. Alternatively, PGC-1 mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.(1993) Science 261:1411-1418.

[0080] Alternatively, PGC-1 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe PGC-1 (e.g., the PGC-1 promoter and/or enhancers) to form triplehelical structures that prevent transcription of the PGC-1 gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N. Y Acad. Sc. 660:27-36; andMaher, L. J. (1992) Bioassays 14(12):807-15.

[0081] II: Recombinant Expression Vectors and Host Cells

[0082] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding PGC-1 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0083] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The “regulatorysequence” is intended to includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., PGC-1proteins, mutant forms of PGC-1, fusion proteins, etc.).

[0084] The recombinant expression vectors of the invention can bedesigned for expression of PGC-1 in prokaryotic or eukaryotic cells. Forexample, PGC-1 can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

[0085] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:3140), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the PGC-1 is cloned into a pGEXexpression vector to create a vector encoding a ftsion proteincomprising, from the N-terminus to the C-terrrinus, GST-thrombincleavage site-PGC-1. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PGC-1unfused to GST can be recovered by cleavage of the fusion protein withthrombin.

[0086] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89) Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gnl). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0087] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to alterthe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al. (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0088] In another embodiment, the PGC-1 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),pMFa (Kuijan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

[0089] Alternatively, PGC-1 can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0090] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0091] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund etal. (1985) Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss (1990) Science 249:374-379) and the α-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0092] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PGC-1 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0093] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0094] A host cell can be any prokaryotic or eukaryotic cell. Forexample, PGC-1 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

[0095] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0096] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding PGC-1 or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0097] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) PGC-1protein. Accordingly, the invention further provides methods forproducing PGC-1 protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding PGC-1 has beenintroduced) in a suitable medium until PGC-1 is produced. In anotherembodiment, the method further comprises isolating PGC-1 from the mediumor the host cell.

[0098] The host cells of the invention can also be used to producenonhuman transgenic animals. The nonhuman transgenic animals can be usedin screening assays designed to identify agents or compounds, e.g.,drugs, pharmaceuticals, etc., which are capable of amelioratingdetrimental symptoms of selected disorders such as weight disorders ordisorders associated with insufficient insulin activity. For example, inone embodiment, a host cell of the invention is a fertilized oocyte oran embryonic stem cell into which PGC-1-coding sequences have beenintroduced. Such host cells can then be used to create non-humantransgenic animals in which exogenous PGC-1 sequences have beenintroduced into their genome or homologous recombinant animals in whichendogenous PGC-1 sequences have been altered. Such animals are usefulfor studying the function and/or activity of PGC-1 and for identifyingand/or evaluating modulators of PGC-1 activity. As used herein, a“transgenic animal” is a nonhuman animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous PGC-1 gene has been alteredby homologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

[0099] A transgenic animal of the invention can be created byintroducing PGC-1-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The human PGC-1 cDNA sequence can be introduced as a transgene into thegenome of a nonhuman animal. Alternatively, a nonhuman homologue of thehuman PGC-1 gene (SEQ ID NO:4), such as a mouse PGC-1 gene (SEQ IDNO:1), can used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the PGC-1 transgene to direct expression ofPGC-1 protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the PGC-1 transgene in its genomeand/or expression of PGC-1 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding PGC-1 can further be bred to other transgenic animalscarrying other transgenes.

[0100] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of a PGC-1 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PGC-1 gene. The PGC-1 gene can be a human gene(e.g., from a human genomic clone isolated from a human genomic libraryscreened with the cDNA of SEQ ID NO:1), but more preferably, is anonhuman homologue of a human PGC-1 gene. For example, a mouse PGC-1gene can be used to construct a homologous recombination vector suitablefor altering an endogenous PGC-1 gene in the mouse genome. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous PGC-1 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous PGC-1 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of tlfe endogenous PGC-1 protein). In the homologousrecombination vector, the altered portion of the PGC-1 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the PGC-1 gene toallow for homologous recombination to occur between the exogenous PGC-1gene carried by the vector and an endogenous PGC-1 gene in an embryonicstem cell. The additional flanking PGC-1 nucleic acid is of sufficientlength for successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the vector (see e.g., Thomas, K. R. and Capecchi,M. R. (1987) Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced PGC-1 genehas homologously recombined with the endogenous PGC-1 gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells arethen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

[0101] In another embodiment, transgenic nonhumans animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0102] Clones of the nonhuman transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0103] III. Isolated PGC-1 Proteins and Anti-PGC-1 Antibodies

[0104] Another aspect of the invention pertains to isolated PGC-1proteins, and biologically active portions thereof, as well as peptidefragments suitable for use as immunogens to raise anti-PGC-1 antibodies.An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material when produced byrecombinant DNA techniques, or chemical precursors or other chemicalswhen chemically synthesized. The language “substantially free ofcellular material” includes preparations of PGC-1 protein in which theprotein is separated from cellular components of the cells in which itis naturally or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of PGC-1protein having less than about 30% (by dry weight) of non-PGC-1 protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-PGC-1 protein, still more preferably lessthan about 10% of non-PGC-1 protein, and most preferably less than about5% non-PGC-1 protein. When the PGC-1 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.The language “substantially free of chemical precursors or otherchemicals” includes preparations of PGC-1 protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of PGC-1 protein having less than about 30% (bydry weight) of chemical precursors or non-PGC-1 chemicals, morepreferably less than about 20% chemical precursors or non-PGC-1chemicals, still more preferably less than about 10% chemical precursorsor non-PGC-1 chemicals, and most preferably less than about 5% chemicalprecursors or non-PGC-1 chemicals. In preferred embodiments, isolatedproteins or biologically active portions thereof lack contaminatingproteins from the same animal from which the PGC-1 protein is derived.Typically, such proteins are produced by recombinant expression of, forexample, a human PGC-1 protein in a nonhuman cell.

[0105] An isolated PGC-1 protein or a portion thereof of the inventionhas one or more of the following biological activities: 1) it caninteract with (e.g., bind to) PPARγ; 2) it can modulate PPARγ activity;3) it can modulate UCP expression; 4) it can modulate thermogenesis inadipocytes, e.g., thermogenesis in brown adipocytes, or muscle; 5) itcan modulate oxygen consumption in adipocytes or muscle; 6) it canmodulate adipogenesis, e.g., differentiation of white adipocytes intobrown adipocytes; 7) it can modulate insulin sensitivity of cells, e.g.,insulin sensitivity of muscle cells, liver cells, adipocytes; 8) it caninteract with (e.g., bind to) nuclear hormone receptors, e.g., thethyroid hormone receptor, the estrogen receptor, the retinoic acidreceptor; 9) it can modulate the activity of nuclear hormone receptors;and 10) it can interact with (e.g., bind to) the transcription factorC/EBPα. In a preferred embodiment, the PGC-1 protein can modulatedifferentiation of white adipocytes to brown adipocytes and/orthermogenesis in brown adipocytes or muscle cells.

[0106] In preferred embodiments, the protein or portion thereofcomprises an amino acid sequence which is sufficiently homologous to anamino acid sequence of SEQ ID NO:2, SEQ ID NO:5 such that the protein orportion thereof maintains the ability to modulate differentiation ofadipocytes and/or thermogenesis in brown adipocytes. The portion of theprotein is preferably a biologically active portion as described herein.In another preferred embodiment, the PGC-1 protein (i.e., amino acidresidues 1-797 and amino acid residues 1-798) has an amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:5, respectively, or an amino acidsequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the amino acid sequence shown inSEQ ID NO:2, SEQ ID NO:5. In yet another preferred embodiment, the PGC-1protein has an amino acid sequence which is encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 or a nucleotidesequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence shown inSEQ ID NO:1, SEQ ID NO:4. The preferred PGC-1 proteins of the presentinvention also preferably possess at least one of the PGC-1 biologicalactivities described herein. For example, a preferred PGC-1 protein ofthe present invention includes an amino acid sequence encoded by anucleotide sequence which hybridizes, e.g., hybridizes under stringentconditions, to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 andwhich can modulate differentiation of white adipocytes to brownadipocytes and/or thermogenesis of brown adipocytes.

[0107] In other embodiments, the PGC-1 protein is substantiallyhomologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 andretains the functional activity of the protein of SEQ ID NO:2, SEQ IDNO:5 yet differs in amino acid sequence due to natural allelic variationor mutagenesis, as described in detail in subsection I above.Accordingly, in another embodiment, the PGC-1 protein is a protein whichcomprises an amino acid sequence which is at least about 50%, preferablyat least about 60%, more preferably at least about 70%, yet morepreferably at least about 80%, still more preferably at least about 90%,and most preferably at least about 95% or more homologous to the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:5.

[0108] Biologically active portions of the PGC-1 protein includepeptides comprising amino acid sequences derived from the amino acidsequence of the PGC-1 protein, e.g., the amino acid sequence shown inSEQ ID NO:2, SEQ ID NO:5 or the amino acid sequence of a proteinhomologous to the PGC-1 protein, which include less amino acids than thefull length PGC-1 protein or the full length protein which is homologousto the PGC-1 protein, and exhibit at least one activity of the PGC-1protein. Typically, biologically active portions (peptides, e.g.,peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39,40, 50, 100 or more amino acids in length) comprise a domain or motif,e.g., a tyrosine phosphorylation site, a cAMP phosphorylation site, aserine-arginine (SR) rich domain, and/or an RNA binding motif, with atleast one activity of the PGC-1 protein. In a preferred embodiment, thebiologically active portion of the protein which includes one or morethe domains/motifs described herein can modulate differentiation ofadipocytes and/or thermogenesis in brown adipocytes. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the activities described herein. Preferably, the biologicallyactive portions of the PGC-1 protein include one or more selecteddomains/motifs or portions thereof having biological activity.

[0109] PGC-1 proteins are preferably produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding the protein iscloned into an expression vector (as described above), the expressionvector is introduced into a host cell (as described above) and the PGC-1protein is expressed in the host cell. The PGC-1 protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Alternative to recombinantexpression, a PGC-1 protein, polypeptide, or peptide can be synthesizedchemically using standard peptide synthesis techniques. Moreover, nativePGC-1 protein can be isolated from cells (e.g., brown adipocytes), forexample using an anti-PGC-1 antibody (described further below).

[0110] The invention also provides PGC-1 chimeric or fusion proteins. Asused herein, a PGC-1 “chimeric protein” or “fusion protein” comprises aPGC-1 polypeptide operatively linked to a non-PGC-1 polypeptide. A“PGC-1 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to PGC-1, whereas a “non-PGC-1 polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the PGC-1 protein,e.g., a protein which is different from the PGC-1 protein and which isderived from the same or a different organism. Within the fusionprotein, the term “operatively linked” is intended to indicate that thePGC-1 polypeptide and the non-PGC-1 polypeptide are fused in-frame toeach other. The non-PGC-1 polypeptide can be fused to the N-terminus orC-terminus of the PGC-1 polypeptide. For example, in one embodiment thefusion protein is a GST-PGC-1 fusion protein in which the PGC-1sequences are fused to the C-terminus of the GST sequences (see ExampleIV). Such fusion proteins can facilitate the purification of recombinantPGC-1. In another embodiment, the fusion protein is a PGC-1 proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofPGC-1 can be increased through use of a heterologous signal sequence.

[0111] Preferably, a PGC-1 chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). APGC-1-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the PGC-1 protein.

[0112] The present invention also pertains to homologues of the PGC-1proteins which function as either a PGC-1 agonist (mimetic) or a PGC-1antagonist. In a preferred embodiment, the PGC-1 agonists andantagonists stimulate or inhibit, respectively, a subset of thebiological activities of the naturally occurring form of the PGC-1protein. Thus, specific biological effects can be elicited by treatmentwith a homologue of limited function. In one embodiment, treatment of asubject with a homologue having a subset of the biological activities ofthe naturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of thePGC-1 protein.

[0113] Homologues of the PGC-1 protein can be generated by mutagenesis,e.g., discrete point mutation or truncation of the PGC-1 protein. Asused herein, the term “homologue” refers to a variant form of the PGC-1protein which acts as an agonist or antagonist of the activity of thePGC-1 protein. An agonist of the PGC-1 protein can retain substantiallythe same, or a subset, of the biological activities of the PGC-1protein. An antagonist of the PGC-1 protein can inhibit one or more ofthe activities of the naturally occurring form of the PGC-1 protein, by,for example, competitively binding to a downstream or upstream member ofthe PGC-1 cascade which includes the PGC-1 protein. Thus, the mammalianPGC-1 protein and homologues thereof of the present invention can be,for example, either positive or negative regulators of adipocytedifferentiation and/or thermogenesis in brown adipocytes.

[0114] In an alternative embodiment, homologues of the PGC-1 protein canbe identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the PGC-1 protein for PGC-1 protein agonist orantagonist activity. In one embodiment, a variegated library of PGC-1variants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof PGC-1 variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential PGC-1 sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of PGC-1 sequencestherein. There are a variety of methods which can be used to producelibraries of potential PGC-1 homologues from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential PGC-1sequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[0115] In addition, libraries of fragments of the PGC-1 protein codingcan be used to generate a variegated population of PGC-1 fragments forscreening and subsequent selection of homologues of a PGC-1 protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a PGC-1 coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PGC-1 protein.

[0116] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of PGC-1homologues. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recrusive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify PGC-1 homologues (Arkin and Yourvan (1992)PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

[0117] An isolated PGC-1 protein, or a portion or fragment thereof, canbe used as an immunogen to generate antibodies that bind PGC-1 usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length PGC-1 protein can be used or, alternatively, theinvention provides antigenic peptide fragments of PGC-1 for use asimmunogens. The antigenic peptide of PGC-1 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:2, SEQ IDNO:5 or a homologous amino acid sequence as described herein andencompasses an epitope of PGC-1 such that an antibody raised against thepeptide forms a specific immune complex with PGC-1. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues. Preferred epitopes encompassed by the antigenic peptide areregions of PGC-1 that are located on the surface of the protein, e.g.,hydrophilic regions.

[0118] A PGC-1 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed PGC-1 protein or achemically synthesized PGC-1 peptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic PGC-1 preparation induces a polyclonal anti-PGC-1antibody response.

[0119] Accordingly, another aspect of the invention pertains toanti-PGC-1 antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as PGC-1. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind PGC-1.The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of PGC-1. A monoclonal antibody compositionthus typically displays a single binding affinity for a particular PGC-1protein with which it immunoreacts.

[0120] Polyclonal anti-PGC-1 antibodies can be prepared as describedabove by immunizing a suitable subject with a PGC-1 immunogen. Theanti-PGC-1 antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized PGC-1. If desired, the antibodymolecules directed against PGC-1 can be isolated from the mammal (e.g.,from the blood) and further purified by well known techniques, such asprotein A chromatography to obtain the IgG fraction. At an appropriatetime after immunization, e.g., when the anti-PGC-1 antibody titers arehighest, antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem 0.255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75),the more recent human B cell hybridoma technique (Kozbor et al. (1983)Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985),Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)or trioma techniques. The technology for producing monoclonal antibodyhybridomas is well known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36).Briefly, an immortal cell line (typically a myeloma) is fused tolymphocytes (typically splenocytes) from a mammal immunized with a PGC-1immunogen as described above, and the culture supernatants of theresulting hybridoma cells are screened to identify a hybridoma producinga monoclonal antibody that binds PGC-1.

[0121] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-PGC-1 monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindPGC-1, e.g., using a standard ELISA assay.

[0122] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-PGC-1 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with PGC-1 to thereby isolateimmunoglobulin library members that bind PGC-1. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:41334137; Barbaset al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature (1990)348:552-554.

[0123] Additionally, recombinant anti-PGC-1 antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)PNAS 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun etal. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc. Res.47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al.(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

[0124] An anti-PGC-1 antibody (e.g., monoclonal antibody) can be used toisolate PGC-1 by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-PGC-1 antibody can facilitate thepurification of natural PGC-1 from cells and of recombinantly producedPGC-1 expressed in host cells. Moreover, an anti-PGC-1 antibody can beused to detect PGC-1 protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the PGC-1 protein. Anti-PGC-1 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I,³⁵S or ³H.

[0125] IV. Pharmaceutical Compositions

[0126] The PGC-1 nucleic acid molecules, PGC-1 proteins, PGC-1modulators, and anti-PGC-1 antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject,e.g., a human. Such compositions typically comprise the nucleic acidmolecule, protein, modulator, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, such media can be used in the compositions of theinvention. Supplementary active compounds can also be incorporated intothe compositions.

[0127] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0128] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0129] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a. PGC-1 protein or anti-PGC-1 antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0130] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipierits and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0131] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0132] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0133] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0134] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0135] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0136] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0137] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0138] V. Uses and Methods of the Invention

[0139] The nucleic acid molecules, polypeptides, polypeptide homologues,modulators, and antibodies described herein can be used in one or moreof the following methods: 1) drug screening assays; 2) diagnosticassays; and 3) methods of treatment. A PGC-1 protein of the inventionhas one or more of the activities described herein and can thus be usedto, for example, modulate adipocyte differentiation, thermogenesis inbrown adipocytes, and insulin sensitivity in various cells, e.g., musclecells, liver cells, and adipocytes. The isolated nucleic acid moleculesof the invention can be used to express PGC-1 protein (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect PGC-1 mRNA (e.g., in a biological sample) or agenetic lesion in a PGC-1 gene, and to modulate PGC-1 activity, asdescribed further below. In addition, the PGC-1 proteins can be used toscreen drugs or compounds which modulate PGC-1 protein activity as wellas to treat disorders characterized by insufficient production of PGC-1protein or production of PGC-1 protein forms which have decreasedactivity compared to wild type PGC-1. Moreover, the anti-PGC-1antibodies of the invention can be used to detect and isolate PGC-1protein and modulate PGC-1 protein activity.

[0140] a. Drug Screening Assays:

[0141] The invention provides methods for identifying compounds oragents which can be used to treat disorders characterized by (orassociated with) aberrant or abnormal PGC-1 nucleic acid expressionand/or PGC-1 polypeptide activity. These methods are also referred toherein as drug screening assays and typically include the step ofscreening a candidate/test compound or agent for the ability to interactwith (e.g., bind to) a PGC-1 protein, to modulate the interaction of aPGC-1 protein and a target molecule, and/or to modulate PGC-1 nucleicacid expression and/or PGC-1 protein activity. Candidate/test compoundsor agents which have one or more of these abilities can be used as drugsto treat disorders characterized by aberrant or abnormal PGC-1 nucleicacid expression and/or PGC-1 protein activity. Candidate/test compoundsinclude, for example, 1) peptides such as soluble peptides, includingIg-tailed fusion peptides and members of random peptide libraries (see,e.g., Lam, K. S. et al. (1991) Nature 354:82-84; Houghten, R. et al.(1991) Nature 354:84-86) and combinatorial chemistry-derived molecularlibraries made of D- and/or L-configuration amino acids; 2)phosphopeptides (e.g., members of random and partially degenerate,directed phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993)Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal,humanized, anti-idiotypic, chimeric, and single chain antibodies as wellas Fab, F(ab′)₂, Fab expression library fragments, and epitope-bindingfragments of antibodies); and 4) small organic and inorganic molecules(e.g., molecules obtained from combinatorial and natural productlibraries).

[0142] In one embodiment, the invention provides assays for screeningcandidate/test compounds which interact with (e.g., bind to) PGC-1protein. Typically, the assays are cell-free assays which include thesteps of combining a PGC-1 protein or a biologically active portionthereof, and a candidate/test compound, e.g., under conditions whichallow for interaction of (e.g., binding of) the candidate/test compoundto the PGC-1 protein or portion thereof to form a complex, and detectingthe formation of a complex, in which the ability of the candidatecompound to interact with (e.g., bind to) the PGC-1 polypeptide orfragment thereof is indicated by the presence of the candidate compoundin the complex. Formation of complexes between the PGC-1 protein and thecandidate compound can be quantitated, for example, using standardimmunoassays.

[0143] In another embodiment, the invention provides screening assays toidentify candidate/test compounds which modulate (e.g., stimulate orinhibit) the interaction (and most likely PGC-1 activity as well)between a PGC-1 protein and a molecule (target molecule) with which thePGC-1 protein normally interacts. Examples of such target moleculesinclude proteins in the same signaling path as the PGC-1 protein, e.g.,proteins which may function upstream (including both stimulators andinhibitors of activity) or downstream of the PGC-1 protein in a pathwayinvolving regulation of body weight, e.g., PPARγ, C/EBPα, nuclearhormone receptors such as the thyroid hormone receptor, the estrogenreceptor, and the retinoic acid receptor, or in a pathway involvinginsulin sensitivity, e.g., PPARγ. Typically, the assays are cell-freeassays which include the steps of combining a PGC-1 protein or abiologically active portion thereof, a PGC-1 target molecule and acandidate/test compound, e.g., under conditions wherein but for thepresence of the candidate compoimd, the PGC-1 protein or biologicallyactive portion thereof interacts with (e.g., binds to) the targetmolecule, and detecting the formation of a complex which includes thePGC-1 protein and the target molecule or detecting theinteraction/reaction of the PGC-1 protein and the target molecule.Detection of complex formation can include direct quantitation of thecomplex by, for example, measuring inductive effects of the PGC-1protein. A statistically significant change, such as a decrease, in theinteraction of the PGC-1 and target molecule (e.g., in the formation ofa complex between the PGC-1 and the target molecule) in the presence ofa candidate compound (relative to what is detected in the absence of thecandidate compound) is indicative of a modulation (e.g., stimulation orinhibition) of the interaction between the PGC-1 protein and the targetmolecule. Modulation of the formation of complexes between the. PGC-1protein and the target molecule can be quantitated using, for example,an immunoassay.

[0144] To perform the above drug screening assays, it is desirable toimmobilize either PGC-1 or its target molecule to facilitate separationof complexes from uncomplexed forms of one or both of the proteins, aswell as to accommodate automation of the assay. Interaction (e.g.,binding of) of PGC-1 to a target molecule, in the presence and absenceof a candidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion polypeptide can be provided which adds a domain that allows thepolypeptide to be bound to a matrix. For example,glutathione-S-transferase/PGC-1 fusion polypeptides can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g. ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofPGC-1-binding polypeptide found in the bead fraction quantitated fromthe gel using standard electrophoretic techniques.

[0145] Other techniques for immobilizing polypeptides on matrices canalso be used in the drug screening assays of the invention. For example,either PGC-1 or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated PGC-1 molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withPGC-1 but which do not interfere with binding of the polypeptide to itstarget molecule can be derivatized to the wells of the plate, and PGC-1trapped in the wells by antibody conjugation. As described above,preparations of a PGC-1-binding polypeptide and a candidate compound areincubated in the PGC-1-presenting wells of the plate, and the amount ofcomplex trapped in the well can be quantitated. Methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the PGC-1 target molecule, or which arereactive with PGC-1 polypeptide and compete with the target molecule; aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0146] In yet another embodiment, the invention provides a method foridentifying a compound (e.g., a screening assay) capable of use in thetreatment of a disorder characterized by (or associated with) aberrantor abnormal PGC-1 nucleic acid expression or PGC-1 polypeptide activity.This method typically includes the step of assaying the ability of thecompound or agent to modulate the expression of the PGC-1 nucleic acidor the activity of the PGC-1 protein thereby identifying a compound fortreating a disorder characterized by aberrant or abnormal PGC-1 nucleicacid expression or PGC-1 polypeptide activity. Disorders characterizedby aberrant or abnormal PGC-1 nucleic acid expression or PGC-1 proteinactivity are described herein. Methods for assaying the ability of thecompound or agent to modulate the expression of the PGC-1 nucleic acidor activity of the PGC-1 protein are typically cell-based assays. Forexample, cells which are sensitive to ligands which transduce signalsvia a pathway involving PGC-1 can be induced to overexpress a PGC-1protein in the presence and absence of a candidate compound. Candidatecompounds which produce a statistically significant change inPGC-1-dependent responses (either stimulation or inhibition) can beidentified. In one embodiment, expressi of the PGC-1 nucleic acid oractivity of a PGC-1 protein is modulated in cells and the effects ofcandidate compounds on the readout of interest (such as rate of cellproliferation or differentiation) are measured. For example, theexpression of genes which are up- or down-regulated in response to aPGC-1 protein-dependent signal cascade can be assayed. In preferredembodiments, the regulatory regions of such genes, e.g., the 5′ flankingpromoter and enhancer regions, are operably linked to a detectablemarker (such as luciferase) which encodes a gene product that can bereadily detected. Phosphorylation of PGC-1 or PGC-1 target molecules canalso be measured, for example, by immunoblotting.

[0147] Alternatively, modulators of PGC-1 nucleic acid expression (e.g.,compounds which can be used to treat a disorder characterized byaberrant or abnormal PGC-1 nucleic acid expression or PGC-1 proteinactivity) can be identified in a method wherein a cell is contacted witha candidate compound and the expression of PGC-1 mRNA or protein in thecell is determined. The level of expression of PGC-1 mRNA or protein inthe presence of the candidate compound is compared to the level ofexpression of PGC-1 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof PGC-1 nucleic acid expression based on this comparison and be used totreat a disorder characterized by aberrant PGC-1 nucleic acidexpression. For example, when expression of PGC-1 mRNA or polypeptide isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of PGC-1 nucleic acid expression.Alternatively, when PGC-1 nucleic acid expression is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor ofPGC-1 nucleic acid expression. The level of PGC-1 nucleic acidexpression in the cells can be determined by methods described hereinfor detecting PGC-1 mRNA or protein.

[0148] In yet another aspect of the invention, the PGC-1 proteins can beused as “bait proteins” in a two-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact withPGC-1 (“PGC-1-binding proteins” or “PGC-1-bp”) and modulate PGC-1protein activity. Such PGC-1-binding proteins are also likely to beinvolved in the propagation of signals by the PGC-1 proteins as, forexample, upstream or downstream elements of the PGC-1 pathway.

[0149] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Bartel, P. et al. “Using the Two-Hybrid System toDetect Protein-Protein Interactions” in Cellular Interactions inDevelopment: A Practical Approach Hartley, D. A. ed. (Oxford UniversityPress, Oxford, 1993) pp. 153-179. Briefly, the assay utilizes twodifferent DNA constructs. In one construct, the gene that codes forPGC-1 is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedpolypeptide (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aPGC-1-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes thepolypeptide which interacts with PGC-1.

[0150] Modulators of PGC-1 protein activity and/or PGC-1 nucleic acidexpression identified according to these drug screening assays can beused to treat, for example, weight disorders, e.g. obesity, anddisorders associated with insufficient insulin activity, e.g., diabetes.These methods of treatment include the steps of administering themodulators of PGC-1 protein activity and/or nucleic acid expression,e.g., in a pharmaceutical composition as described in subsection IVabove, to a subject in need of such treatment, e.g., a subject with adisorder described herein.

[0151] b. Diagnostic Assays:

[0152] The invention further provides a method for detecting thepresence of PGC-1 in a biological sample. The method involves contactingthe biological sample with a compound or an agent capable of detectingPGC-1 polypeptide or mRNA such that the presence of PGC-1 is detected inthe biological sample. A preferred agent for detecting PGC-1 mRNA is alabeled or labelable nucleic acid probe capable of hybridizing to PGC-1mRNA. The nucleic acid probe can be, for example, the full-length PGC-1cDNA of SEQ ID NO:1, or a portion thereof, such as an oligonucleotide ofat least 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to PGC-1mRNA. A preferred agent for detecting PGC-1 protein is a labeled orlabelable antibody capable of binding to PGC-1 protein. Antibodies canbe polyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeledor labelable”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sarnple” is intended to include tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject. That is, the detection method of theinvention can be used to detect PGC-1 mRNA or protein in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of PGC-1 mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of PGC-1 proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, PGC-1protein can be detected in vivo in a subject by introducing into thesubject a labeled anti-PGC-1 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

[0153] The invention also encompasses kits for detecting the presence ofPGC-1 in a biological sample. For example, the kit can comprise alabeled or labelable compound or agent capable of detecting PGC-1protein or mRNA in a biological sample; means for determining the amountof PGC-1 in the sample; and means for comparing the amount of PGC-1 inthe sample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect PGC-1 mRNA or protein.

[0154] The methods of the invention can also be used to detect geneticlesions in a PGC-1 gene, thereby determining if a subject with thelesioned gene is at risk for a disorder characterized by aberrant orabnormal PGC-1 nucleic acid expression or PGC-1 protein activity asdefined herein. In preferred embodiments, the methods include detecting,in a sample of cells from the subject, the presence or absence of agenetic lesion characterized by at least one of an alteration affectingthe integrity of a gene encoding a PGC-1 protein, or the misexpressionof the PGC-1 gene. For example, such genetic lesions can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a PGC-1 gene; 2) an addition of one or morenucleotides to a PGC-1 gene; 3) a substitution of one or morenucleotides of a PGC-1 gene, 4) a chromosomal rearrangement of a PGC-1gene; 5) an alteration in the level of a messenger RNA transcript of aPGC-1 gene, 6) aberrant modification of a PGC-1 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a PGC-1 gene, 8)a non-wild type level of a PGC-1-protein, 9) allelic loss of a PGC-1gene, and 10) inappropriate post-translational modification of aPGC-1-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in aPGC-1 gene.

[0155] In certain embodiments, detection of the lesion involves the useof a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the PGC-1-gene (see Abravaya et al. (1995)Nucleic Acids Res 0.23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a PGC-1 gene under conditions such that hybridization andamplification of the PGC-1-gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample.

[0156] In an alternative embodiment, mutations in a PGC-1 gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0157] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the PGC-1gene and detect mutations by comparing the sequence of the sample PGC-1with the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463).A variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

[0158] Other methods for detecting mutations in the PGC-1 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985)Science 230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al.(1992) Meth. Enzymol. 217:286-295), electrophoretic mobility of mutantand wild type nucleic acid is compared (Orita et al. (1989) PNAS86:2766; Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79), and movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (Myers et al (1985) Nature313:495). Examples of other techniques for detecting point mutationsinclude, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

[0159] c. Methods of Treatment

[0160] Another aspect of the invention pertains to methods for treatinga subject, e.g., a human, having a disease or disorder characterized by(or associated with) aberrant or abnormal PGC-1 nucleic acid expressionand/or PGC-1 protein activity. These methods include the step ofadministering a PGC-1 modulator to the subject such that treatmentoccurs. The language “aberrant or abnormal PGC-1 expression” refers toexpression of a non-wild-type PGC-1 protein or a non-wild-type level ofexpression of a PGC-1 protein. Aberrant or abnormal PGC-1 proteinactivity refers to a non-wild-type PGC-1 protein activity or anon-wild-type level of PGC-1 protein activity. As the PGC-1 protein isinvolved in, for example, a pathway involving adipocyte differentiation,thermogenesis in brown adipocytes, and insulin sensitivity, aberrant orabnormal PGC-1 protein activity or nucleic acid expression interfereswith the normal weight control and metabolic functions. Non-limitingexamples of disorders or diseases characterized by or associated withabnormal or aberrant PGC-1 protein activity or nucleic acid expressioninclude weight disorders, e.g., obesity, cachexia, anorexia, anddisorders associated with insufficient insulin activity, e.g., diabetes.Disorders associated with body weight are disorders associated withabnormal body weight or abnormal control of body weight. As used herein,the language “diseases associated with or characterized by insufficientinsulin activity” include disorders or diseases in which there is anabnormal utilization of glucose due to abnormal insulin function.Abnormal insulin function includes any abnormality or impairment ininsulin production, e.g., expression and/or transport through cellularorganelles, such as insulin deficiency resulting from, for example, lossof P cells as in IDDM (Type I diabetes), secretion, such as impairmentof insulin secretory responses as in NIDDM (Type II diabetes), the formof the insulin molecule itself, e.g., primary, secondary or tertiarystructure, effects of insulin on target cells, e.g., insulin-resistancein bodily tissues, e.g., peripheral tissues, and responses of targetcells to insulin. See Braunwald, E. et al. eds. Harrison's Principles ofInternal Medicine, Eleventh Edition (McGraw-Hill Book Company, New York,1987) pp. 1778-1797; Robbins, S. L. et al. Pathologic Basis of Disease,3rd Edition (W.B. Saunders Company, Philadelphia, 1984) p. 972 forfurther descriptions of abnormal insulin activity in IDDM and NIDDM andother forms of diabetes. The terms “treating” or “treatment”, as usedherein, refer to reduction or alleviation of at least one adverse effector symptom of a disorder or disease, e.g., a disorder or diseasecharacterized by or associated with abnormal or aberrant PGC-1 proteinactivity or PGC-1 nucleic acid expression.

[0161] As used herein, a PGC-1 modulator is a molecule which canmodulate PGC-1 nucleic acid expression and/or PGC-1 protein activity.For example, a PGC-1 modulator can modulate, e.g., upregulate (activate)or downregulate (suppress), PGC-1 nucleic acid expression. In anotherexample, a PGC-1 modulator can modulate (e.g., stimulate or inhibit)PGC-1 protein activity. If it is desirable to treat a disorder ordisease characterized by (or associated with) aberrant or abnormal(non-wild-type) PGC-1 nucleic acid expression and/or PGC-1 proteinactivity by inhibiting PGC-1 nucleic acid expression, a PGC-1 modulatorcan be an antisense molecule, e.g., a ribozyme, as described herein.Examples of antisense molecules which can be used to inhibit PGC-1nucleic acid expression include antisense molecules which arecomplementary to a portion of the 5′ untranslated region of SEQ ID NO:1,SEQ ID NO:4 which also includes the start codon and antisense moleculeswhich are complementary to a portion of the 3′ untranslated region ofSEQ ID NO:1, SEQ ID NO:4. A PGC-1 modulator which inhibits PGC-1 nucleicacid expression can also be a small molecule or other drug, e.g., asmall molecule or drug identified using the screening assays describedherein, which inhibits PGC-1 nucleic acid expression. If it is desirableto treat a disease or disorder characterized by (or associated with)aberrant or abnormal (non-wild-type) PGC-1 nucleic acid expressionand/or PGC-1 protein activity by stimulating PGC-1 nucleic acidexpression, a PGC-1 modulator can be, for example, a nucleic acidmolecule encoding PGC-1 (e.g., a nucleic acid molecule comprising anucleotide sequence homologous to the nucleotide sequence of SEQ IDNO:1, SEQ ID NO:4) or a small molecule or other drug, e.g., a smallmolecule (peptide) or drug identified using the screening assaysdescribed herein, which stimulates PGC-1 nucleic acid expression.

[0162] Alternatively, if it is desirable to treat a disease or disordercharacterized by (or associated with) aberrant or abnormal(non-wild-type) PGC-1 nucleic acid expression and/or PGC-1 proteinactivity by inhibiting PGC-1 protein activity, a PGC-1 modulator can bean anti-PGC-1 antibody or a small molecule or other drug, e.g., a smallmolecule or drug identified using the screening assays described herein,which inhibits PGC-1 protein activity. If it is desirable to treat adisease or disorder characterized by (or associated with) aberrant orabnormal (non-wild-type) PGC-1 nucleic acid expression and/or PGC-1protein activity by stimulating PGC-1 protein activity, a PGC-1modulator can be an active PGC-1 protein or portion thereof (e.g., aPGC-1 protein or portion thereof having an amino acid sequence which ishomologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5 or aportion thereof) or a small molecule or other drug, e.g., a smallmolecule or drug identified using the screening assays described herein,which stimulates PGC-1 protein activity.

[0163] In addition, a subject having a weight disorder, e.g., obesity,can be treated according to the present invention by administering tothe subject a PGC-1 protein or portion thereof or a nucleic acidencoding a PGC-1 protein or portion thereof such that treatment occurs.Similarly, a subject having a disorder associated with insufficientinsulin activity can be treated according to the present invention byadministering to the subject a PGC-1 protein or portion thereof or anucleic acid encoding a PGC-1 protein or portion thereof such thattreatment occurs.

[0164] Other aspects of the invention pertain to methods for modulatinga cell associated activity. These methods include contacting the cellwith an agent (or a composition which includes an effective amount of anagent) which modulates PGC-1 protein activity or PGC-1 nucleic acidexpression such that a cell associated activity is altered relative to acell associated activity of the cell in the absence of the agent. Asused herein, “a cell associated activity” refers to a normal or abnormalactivity or function of a cell. Examples of cell associated activitiesinclude proliferation, migration, differentiation, production orsecretion of molecules, such as proteins, cell survival, andthermogenesis. In a preferred embodiment, the cell associated activityis thermogenesis and the cell is a brown adipocyte. The term “altered”as used herein refers to a change, e.g., an increase or decrease, of acell associated activity. In one embodiment, the agent stimulates PGC-1protein activity or PGC-1 nucleic acid expression. Examples of suchstimulatory agents include an active PGC-1 protein, a nucleic acidmolecule encoding PGC-1 that has been introduced into the cell, and amodulatory agent which stimulates PGC-1 protein activity or PGC-1nucleic acid expression and which is identified using the drug screeningassays described herein. In another embodiment, the agent inhibits PGC-1protein activity or PGC-1 nucleic acid expression. Examples of suchinhibitory agents include an antisense PGC-1 nucleic acid molecule, ananti-PGC-1 antibody, and a modulatory agent which inhibits PGC-1 proteinactivity or PGC-1 nucleic acid expression and which is identified usingthe drug screening assays described herein. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).In a preferred embodiment, the modulatory methods are performed in vivo,i.e., the cell is present within a subject, e.g., a mammal, e.g., ahuman, and the subject has a disorder or disease characterized by orassociated with abnormal or aberrant PGC-1 protein activity or PGC-1nucleic acid expression.

[0165] A nucleic acid molecule, a protein, a PGC-1 modulator, a compoundetc. used in the methods of treatment can be incorporated into anappropriate pharmaceutical composition described herein and administeredto the subject through a route which allows the molecule, protein,modulator, or compound etc. to perform its intended function. Examplesof routes of administration are also described herein under subsectionIV.

[0166] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patent applications, patents, and published patentapplications cited throughout this application are hereby incorporatedby reference.

EXAMPLES Example I Identification and Characterization of Mouse PGC-1

[0167] The mouse HIB zB cell line (Ross, R. et al. (1992) PNAS89:7561-7565), a brown adipocyte cell line which expresses UCP, wasdifferentiated and treated with isoproterenol to induce UCP expression.A cDNA library from the mouse HIB 1B cell line was screened in a yeasttwo hybrid system using PPARγ as bait and Clontech (Palo Alto, Calif.)reagents. Briefly, amino acids 183-505 of the murine PPARγ were clonedin-frame into the GAL4 DNA-binding domain plasmid pAS2. A HIB 1B cDNAexpression library was constructed in the GAL4 activation domain plasmidpACT II. Yeast two-hybrid system protocol was described as as describedin the CLONTECH Matchmaker two-hybrid system protocol. pAS-PPARγ wastransformed into Y190 yeast cells by the lithium acetate method andmaintained by selection in leucine-plates. A pACT-HIB 1B cDNA librarywas transformed into Y190-PPARγ yeast cells, and positive clones wereassayed for β-galactosidase activity in a filter assay as described inthe CLONTECH protocol. pAS1 lamin cDNA was used to obtain fill-lengthPGC-1, the positive yeast cDNA clone was used as probe to screen anoligo dT λZAP cDNA library from HIB 1B cells.

[0168] A screen of 1×10⁶ primary transformants using cDNAs prepared fromHIB 1B brown fat cells yielded about 130 clones. The cDNA inserts ofpositive phage clones were excised into pBluescript and both strandswere sequenced by standard methods. These were then analyzed forpreferential expression in brown versus white fat with RNA blots. One ofthe clones obtained using this yeast two hybrid system was a partialPGC-1 clone which comprised nucleotides 610 to 3066 of SEQ ID NO:1. Thefull length clone was obtained by using a partial PGC-1 clone comprisingnucleotides 650 to 3066 of SEQ ID NO:1, to screen a λZAP-HIB 1B library.

[0169] PGC-1 was then subcloned from a PBS plasmid to a PSV.sport (GIBCOBRL, Gaithersburg, Md.) and in vitro translated using the TnT Promegakit (Promega, Madison, Wis.). Two bands were observed in the in vitrotranslated PSV.sport PGC-1 which corresponded to the molecular weightsof about 120 kD and 70 kD. These bands most likely represent differentisoforms of PGC-1. The 120 kD form most likely represents the protein ofSEQ ID NO:2.

[0170] The nucleotide sequence of murine PGC-1 (shown in FIGS. 1A, 1A-1,and 1A-2 and SEQ ID NO:1) includes 3066 nucleotides which encode aprotein containing 797 amino acid residues with a predicted molecularmass of 92 kDa (FIG. 2A). The murine PGC-1 protein sequence (shown inFIGS. 1A, 1A-1, 1A-2, and 2A and SEQ ID NO:2) has several domains/motifsincluding Databank searches indicate that murine PGC-1 represents anovel protein with no close homologs in any databases except expressedsequence tag (EST) databases. It does, however, contain recognizablepeptide motifs including: a putative RNA-binding motif (amino acids677-709) and two so-called SR domains, regions that are rich in serineand arginine residues (amino acids 565-598 and 617-631). Proteinscontaining paired RNA-binding motifs and SR domains have been shown tointeract with the C-terminal domain (CTD) of RNA polymerase II (Yuryevet al. (1996) Proc. Natl. Acad. Sci. USA 93:6975-6980). Except for thesetwo regions, however, PGC-1 shares no other sequence similarity withother proteins that contain these domains. In addition to these domains,PGC-1 also contains three consensus sites for phosphorylation by proteinkinase A. However, no significant homology was discovered between PGC-1and any known coactivator of nuclear receptors. PGC-1 does, however,contain one LXXLL motif (amino acids 142-146), recently identified as anelement that can mediate nuclear receptor-coactivator interactions(Heery et al. (1997) Nature 397:733-736; Torchia et al. (1997) Nature387:677-684).

[0171] From these experiments, it is clear that PGC-1 is a new factorthat interacts with the adipogenic transcription factor PPARγ. Moreover,as it is known that ligands of PPARγ can induce the specific brownadipose tissue marker UCP, PPARγ is believed to play an important rolein brown adipose tissue differentiation. Thus, PGC-1 modulation of PPARγactivity plays a role in brown adipose tissue differentiation, e.g., itcan promote cells to differentiate into brown adipose cells rather thanwhite adipose cells.

Example II Identification of Human PGC-1

[0172] Northern blot analysis of human poly A RNA screened with a mousefull length cDNA probe (e.g., a probe having the sequence shown in SEQID NO:1) revealed high levels of expression of PGC-1 in human muscle,heart, brain, kidney and pancreas, with the highest levels of expressiondetected in human muscle and heart. Accordingly, a human muscle library,e.g., an human skeletal muscle oligo dT primed library (Clontech catalog#HL5023t, lot #7110299), was screened using a full length mouse PGC-1probe comprising the nucleotide sequence of SEQ ID NO:1 or a portionthereof (e.g., nucleotides from the 5′ region of SEQ ID NO:1, e.g.,nucleotides 1-50 of SEQ ID NO:1) (e.g., as described in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). Several overlapping clones wereisolated and sequenced. After several rounds of screening, the longestclone isolated (clone #1¹) contained a fragment with homology startingat amino acid 507 of the mouse sequence in SEQ ID NO:1.

[0173] Using a “5′ race strategy” the full length cDNA sequence wasobtained. A human PGC-1 cDNA clone was obtained with the Marathon RACEprotocol and reagents available commercially through ClontechLaboratories, Inc. RACE, or rapid amplification of cDNA ends, is usefulto isolate a PCR fragment comprising the native 3′ or 5′ end of a cDNAopen reading frame, and involves use of one or more gene-specific sense(for 3′ RACE) or antisense (for 5′ RACE) oligonucleotide primers. TheRACE protocol used is generally as described in Siebert et al. (1995),23 Nucl. Acids Res. 1087-1088, and in the Clontech, Inc. User Manual forMarathon-Ready cDNA (1996), the teachings of which are incorporatedherein by reference. The RACE reagents included the Advantage KlenTaqPolymerase mix, 10× PCR reaction buffer, 50× dNTP mix and Tricine-EDTAbuffer commercially available from Clontech, Inc. The protocol ispracticed with 0.5 mL PCR reaction tubes and a thermal cycling devicesuch as the DNA Thermal Cycler 480 available from Perkin-ElmerCorporation.

[0174] A PGC-1 specific primer (ATCTTCGCTGTCATCAAACAGGCCATC (SEQ IDNO:6)), 27 bp (base-pairs) in length, was prepared for use in a 5′-RACEprotocol to amplify a PCR product comprising the 5′ end of human PGC-1open reading frame in a Human Skeletal Muscle Marathon-Ready cDNApreparation (Clontech catalog #7413-1, lot #8030061). Thermal cyclingwas carried out according to the manufacturer's recommended Program 1 (a94° C. hot start followed by 5 cycles at 94° C. to 72° C., then 5 cyclesat 94° C. to 70° C., then 20-25 cycles at 94° C. to 68° C.).Confirmation that additional human PGC-1 gene sequence has been obtainedcan be produced by routine Southern blot analysis or by subcloning andsequencing. This fragment contained a sequence with homology extendingto the most 5′ sequence of mouse PGC-1 (SEQ ID NO:1). Both clone #1¹ andthe 5′ RACE product were then sequenced on both strands and the fulllength human cDNA sequence (SEQ ID NO:4) was constituted.

[0175] The nucleotide sequence of human PGC-1 (shown in FIG. 7 and SEQID NO:4) includes 3023 nucleotides which encode a protein containing 798amino acid residues with a predicted molecular mass of 92 kDa. The humanPGC-1 protein sequence (shown in FIG. 8 and SEQ ID NO:5) has severaldomains/motifs including Databank searches indicate that human PGC-1represents a novel protein with no close homologs in any databasesexcept expressed sequence tag (EST) databases.

[0176] An alignment between human PGC-1 (SEQ ID NO:5) and mouse PGC-1(SEQ ID NO:2) amino acid sequences was performed using the BLASTsoftware found at the National Center for Biotechnology Information(NCBI) web site (URL: http://www.ncbi.nlm.nih.gov, Altschul, S. F. etal. (1990) J Mol Biol 215:403-410; Madden, T. L. et al. (1996) MethEnzymol 266:131-141) and it was determined that human PGC-1 has a 94%identity to mouse PGC-1.

Example III Tissue Distribution of Mouse PGC-1 and Cold Induction ofPGC-1 in Brown Adipose Tissue

[0177] A Northern analysis of mRNA from lung, muscle, liver, heart,kidney, white adipose tissue (WAT), brown adipose tissue (BAT), brain,testis, and spleen tissue from 4 week old mice acclimated at 24° C.using a probe comprising nucleotides 150 to 3066 of SEQ ID NO:1 wasperformed. Briefly, total RNA was isolated from cultured cells andtissues of mouse by guanidine isothiocyanate extraction. RNA sampleswere processed as previously described (Tontonoz et al. (1994) GenesDev. 8:1224-1234). Three bands appeared on the Northern blots that werelarger than the 28S (5000-6000 bp) marker. These bands most likelyrepresent different isoforms of PGC-1. PGC-1 mRNA was detectedpredominantly in brain, heart, kidney, and BAT. In addition, a minorspecies of approximately 8 kb is also observed in all of these tissues.In contrast, no PGC-1 mRNA expression is observed from white fat, lung,skeletal muscle, liver, testes, or spleen.

[0178] Exposure to cold is a classical inducer of adaptivethermogenesis, especially in brown fat and skeletal muscle (Himms-Hagen(1989) Can. J. Physiol. Pharmcol. 67:394-401). A second Northernanalysis of mRNA from WAT, BAT, and liver tissue from 4 week old miceacclimated at 4° C. from 3 to 12 hours using the same probe as in thefirst Northern analysis was performed. From this Northern analysis, itwas apparent that PGC-1 was highly induced (about 30- to 50-fold) duringcold exposure especially in BAT and that PGC-1 expression was BATspecific with no expression in WAT. Although PGC-1 mRNA expression isnot detectable in skeletal muscle from mice kept at ambient temperature,exposure of mice to cold for 12 hr induces expression of the PGC-1 genein this tissue. Heart and kidney, which express PGC-1 mRNA at roomtemperature, do not elevate this expression upon cold exposure. PGC-1induction during cold exposure parallels that of UCP, a brown fatspecific marker responsible for the thermogenic activity of BAT.

[0179] These experiments show that although PGC-1 is expressed inseveral tissues, including BAT, from animals acclimated to 24° C., it isnot expressed in WAT. The animal studies described herein were carriedout as follows. Four-week-old male C57BL/6J mice were used. Animals werefed ad libitum and 10 animals were grouped per cage. A control group waskept at 24° C., while experimental groups were kept at 4° C for 3 or 12hrs. Animals were sacrified, tissues were dissected and collectedimmediately.

[0180] WAT and BAT share the same genetic and biochemical machinery foradipogenesis except that BAT develops a thermogenic function uponterminal differentiation. Thus, PGC-1 plays a role in the thermogenicfunction of BAT. This function was confirmed when the second Northernanalysis revealed that in tissues from animals acclimated at 4° C.,PGC-1 was expressed essentially only in BAT. PGC-1, therefore, plays arole in the equilibrium between energy storage and expenditure.

[0181] Northern blot mRNA analysis of PGC-1 and genes of mitochondrialfunction in different mouse tissues (kidney, heart, BAT and WAT) aftercold exposure revealed that cold-induced expression of PGC-1 in thebrown fat of these mice correlated with the induced expression of otherkey mitochondrial proteins including ATP-synthetase (β subunit) andcytochrome c-oxidase subunits (COX II and COX IV). Although chronic coldexposure has been reported to lead to elevated activities for thesemitochondrial proteins in skeletal muscle (Bourhim et al. (1990) Am J.Physiol. 258:R1291-R1298), no induction of mRNA for ATP-synthetase, COXII or COX IV was seen in muscle with the relatively brief exposure tocold. To conduct these experiments, animal were maintained at 4° C. for3 or 12 hours, sacrificed and tissues (kidney, heart, WAT and BAT) weredissected for the preparation of RNA. Ten mice were pooled for eachsample. Probes used for hybridization were PGC-1, UCT-1, ACT sythetase(β subunit), cytochrome c-oxidase II (COX-II), and cytochrome c-oxidaseIV (COX-IV).

[0182] Cold is sensed in the central nervous system and results inincreased sympathetic output to peripheral tissues, including muscle andbrown fat Himms-Hagen (1989) Can. J. Physiol. Pharmcol. 67:394-401).Cold exposure can be mimicked, in terms of brown adipocyte precursorcell growth and the induction of UCP-1, by exposure of cultured brownfat cells to β-adrenergic agonists (Rehnmark et al. (1990) J. Biol.Chem. 25:16464-16471). To determine if PGC-1 gene expression is alsosensitive to β-adrenergic agonists, HIB 1B brown fat cells were treatedwith isoproterenol (1 μM), a nonsubtype selective β agonist, for 10 hr.Total cellular RNA was isolated and analyzed using PGC-1 and UCP-1 cDNAprobes.

[0183] Treatment of HIB 1B brown fat cells with these agents resulted ina sharp increase in both PGC-1 mRNA and UCP-1 mRNA. Briefly, HIB 1Bbrown fat preadipocytes were differentiated as described herein. After 6days, cells were approximately 80% differentiated. Exposure of brown fatcells to 9-cis retinoic acid has previously been shown to potentiate theeffects of β agonists to induce UCP-1 expression (Puigserver et al.(1996) Biochem. J. 317:827-833). Addition of this retinoid (whichactivates both RXR and RAR) and isoproterenol to the HIB 1B cellsresulted in a small, further increase in both PGC-1 and UCP-1expression. These results indicate that β-adrenergic agonists may playan important role in mediating the effects of cold on the induction ofboth UCP-1 and PGC-1.

Example IV Recombinant Expression of PGC-1 and Binding of PGC-1 to OtherTranscription Factors and Nuclear Hormone Receptors

[0184] GST-PGC-1 fusion proteins were generated by first subcloning aportion of the PGC-1 nucleotide sequence (nucleotides 610 to 3066 of SEQID NO:1) into a pGEX vector (Pharmacia Biotech Inc., Piscataway, N.J.).Briefly, PGC-1 (EcoRI-Xhol fragment from pBluescript) was cloned intothe Smal site of pGEX 5X3. The PPARγ deletions were generated byperforming PCR using specific oligonucleotides and there were clonedin-frame in pGEX 5X2. These fusion proteins were expressed, and purifiedfrom E. coli on beads containing approximately 1 μg of protein (eitherGST, or alone, or fused to PGC-1), 30 μl was resuspended i the bindingbuffer (20 mM HEPES [pH 7.7], 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2,0.05% NP40, 2 mM DTT, 10% glycerol).

[0185] After expressing the fusion protein in COS cells, in vitrobinding assays as described in Takeshita, A. et al. ((1996)Endocrinology 137:3594-3597) were performed to study the interaction ofPGC-1 with PPARγ, other PPAR isoforms such as PPARα and PPARδ, othertranscription factors such as C/EBPα and RXRα, and other nuclear hormonereceptors such as the thyroid hormone receptor, the estrogen receptor,and the retinoic acid receptor. The assays were carried out as follows.Control GST protein alone or PGC-1 (aa 36-797) fused to GST wereimmobiled on glutathione agarose beads and incubated with different invitro-translated ([³⁵S]methioniné-labeled nuclear receptors andappropriate ligands or vehicle. The fusion proteins were mixed with 5 μlof different nuclear receptors made in an in vitro reticulocytetranslation reaction using [³⁵S] methionine (Promega TNT reticulocytelysate system kit). Specific nuclear receptor ligands or vehicle (5 μl)was added. Binding was performed for 60 min. at room temperature. Thebeads were then washed four times with the binding buffer with orwithout ligands and resuspended in SDS-PAGE sample buffer. Afterelectrophoresis, fixation, and enhancement, the radiolabeled proteinswere visualized by autoradiography.

[0186] These assays show that PGC-1 interacted with PPARγ. Thisinteraction was not ligand-dependent, in that addition of BRL49653 (athiazolidinedione ligand for PPARγ) at 10 μM does not significantlyalter this binding. A similar lack of ligand dependence for thisinteraction was seen when bacterially expressed PPARγ was immobilized onbeads and used with reticulocyte-translated PGC-1. These assays alsoshowed that PGC-1 interacts with: a) PPARα and shows a slight liganddependency using leukotriene-4 (1 μM); b) PPARδ and shows a slightligand dependency using carboprostacyclin (1 μM); c) the thyroid hormonereceptor with a slight ligand dependency using thyroid hormone (1 μM);d) the estrogen receptor with a slight ligand dependency using estradiol(1 μM); and 3) the retinoic acid receptor with a strong liganddependency using all-trans retinoic acid (1 μM). The TRβ also bindsspecifically to PGC-1, though in this case ligand (T₃) addition causes a2- to 3-fold increase in binding. A strong ligand-dependent binding isseen between PGC-1 and the retinoic acid (RA) receptor, and betweenPGC-1 and the estrogen receptor (ERα). In contrast, little or no bindingis seen between PGC-1 and the retinoid-X receptor (RXRα), with orwithout ligand addition. These data indicate that PGC-1 interactsspecifically with PPARγ and several other nuclear receptors in vitro.There is a broad range of dependence on ligand for these interactions,from no ligand dependence (PPARγ) to a strong dependence on ligandaddition (RARα).

[0187] The interaction between PPARγ and PGC-1 can also be seen inmammalian cells. Even in the absence of added ligand, an association isobserved between these two proteins in immunoprecipitation assays.Vectors expressing HA-tagged PGC-1 and PPARγ were transfected into COScells. In brief, full-length PGC-1 with an HA-tagged N terminus wasgenerated by PCR and closed into Smal of pSV-SPORT. Ligands pioglitazone(5 μM), 9-cis RA (1 μM), and 8-Br-cAMP (1 nm) were added 3 hrs. beforecells were harvested. Cell extracts and immunoprecipitation fromtransfected cells were performed as the Lasser et al. ((1991) Cell66:305-315). Rabbit anti-murine PPARγ (Hu et al. (1996) Science274:2100-2103) was used as a 1:500 dilution for immunoprecipitation. Ananti-HA mouse dilution for Western blot that was developed using ECL(Amersham). When cells are treated with pioglitazone (a PPARγ ligand), avery modest increase in association is observed.

[0188] To address whether PGC-1 does indeed reside in the cell nucleus,a fusion protein between PGC-1 and green fluorescent protein (GFP) wasconstructed. GFP fused to the full-length PGC-1 was generated by closingthis (Clontech). Cellular localization was visualized 24 hrs. aftertransfection using a Nikon Diaphora 200 microscope. When GFP-PGC-1 isexpressed in COS cells, it is observed entirely in the cell nucleus.

[0189] These results show that PGC-1 binds not only to PPARγ but also toother nuclear hormone receptors, and thus this molecule can be used tomodulate the function of these additional nuclear hormone receptors.PGC-1 can be used as a target for screening molecules which modulate thefunction of these nuclear hormone receptors. Moreover, the fact thatPGC-1 interacts with the thyroid hormone receptor and the retinoic acidreceptor is important in brown adipocyte function as both of thesereceptors can transcriptionally regulate UCP expression.

Example V PGC-1 Acts as a Coactivator with PPARγ/RXRα and TR to InduceExpression of a Gene Under the Control of UCP Regulatory Elements

[0190] To assess the transcription activity of PGC-1, an in vitrotransciptional assay was performed. The UCP-1 promoter has been shown tohave binding sites for both PPARγ and the TR (Cassard-Doulcier et al.(1994) J. Biol. Chem. 269:24335-24342; Sears et al. (1996) Mol. Cell.Biol. 16:3410-3419). In this assay, the full length promoter andenhancer of UCP was linked to the CAT reporter gene. RAT IR (a ratfibroblast cell line transformed to express the human insulin receptor)cells were transiently transfected with PSV-sport alone (control),PPARγ/RXRα, PGC-1, and PPARγ/RXRα/PGC-1 using the calcium phosphatemethod. Results from CAT assays were controlled for transfectionefficiency by cotransfection of a β-galactosidase reporter gene underthe control of the CMV promoter. In each case, the cells were treatedwith either dimethyl sulfoxide or a combination of 9-cis retinoic acid,8-Br-cAMP, and the synthetic PPARγ ligand pioglitazone (PIO).Transcriptional activity was seen in the cells treated with thecombination of 9-cis retinoic acid, 8-Br-cAMP, and PIO and containingPGC-1 alone, cells containing PPARγ/RXRα, and cells containingPPARγ/RXRα/PGC-1. Maximum activity was seen in cells treated with thecombination of 9-cis retinoic acid, 8-Br-cAMP, and PIO and containingPPARγ/RXRα/PGC-1. These results indicate that PGC-1 acts as a positivetranscriptional coactivator of PPARγ/RXRα.

[0191] To determine which inducers were involved in the transcriptionalactivation of PGC-1, the cells were treated individually (PIO, thesynthetic PPARγ ligand troglitazone (TRO), 9-cis retinoic acid, 8-BrcAMP) and in combination (9-cis retinoic acid in combination with 8-BrcAMP) with different inducers. With regard to the cells treated with theindividual inducers, it was found that the potency of the inducers wasas follows (from highest to lowest): 9-cis retinoic acid, 8-Br cAMP,TRO, and then PIO. The combination of 9-cis retinoic acid and 8-Br cAMPwas more potent in enhancing transcriptional activity than any of theindividual inducers.

[0192] Similarly, TRβ/RXRα combination alone induced very littletranscriptional activity, even when stimulated with a ligand cocktailincluding T₃ (1 μM). However, the combination of PGC-1 with the TRβ/RXRαpair induced powerful transactivation, again in a ligand-dependentmanner. These results clearly indicate that PGC-1 can function as apotent transcriptional coactivator for PPARγ and the TR. It isinteresting that the optimal transcriptional response is seen with PPARγligand is added, despite the fact that the binding of PGC-1 and PPARγ isnot ligand dependent. It is likely that this results from simultaneous,ligand-dependent docking of another coactivator, such as SRC-1, CBP, orothers.

[0193] The role of different hormones and ligands used to achievemaximal transcriptional activation with PPARγ and PGC-1 is dissected inFIG. 3A. The individual components—troglitazone (trog.), 9 cis-retinoicacid (9cRA), and 8-bromo cyclic AMP (cAMP)—each stimulate a 2- to 4-foldincrease in transcriptional activity. The fold activation was comparedto the value observed in cells transfected with the same vectors but nottreated with ligand. However, the most robust responses are seen whenthey are used in combination. The synergistic effect of 9-cis retinoicacid and 8-bromo cyclic AMP is particularly striking (14-fold), whileall three agents together cause an 18-fold increase above the untreatedcontrol.

[0194] The above-described transcriptional assay represents a usefulassay for screening compounds or agents which can modulate, e.g.,stimulate or inhibit, the function of PGC-1 alone and/or PGC-1 incombination with PPARγ/RXRα. Based on the results reported in thisExample, agents which likely modulate UCP expression and thusthermogenesis in BAT include PGC-1 molecules, PPARγ ligands (e.g.,thiazolidinediones, e.g., PIO and TRO), retinoids, and adrenergicagonists.

Example VI Identification of the Domains That Mediate the PGC-1-PPARγInteraction

[0195] The interaction between nuclear receptors and certaincoactivators such as SRC-1 or CBP is ligand dependent (Kamai et al.(1996) Cell 84:403-414) and involves an LXXLL (SEQ ID NO:3) motif in thecoactivators and the C-terminal AF-2 domain in the receptors (Heery etal. (1997) Nature 387:733-736; Torchia et al. (1997) Nature387:677-684). To identify the domains responsible for PGC-1-PPARγinteractions, different C-terminal deletions of PGC-1 were generated asreticulocyte translation products and mixed with a FST-PPARγ fusionprotein. Deletions of PGC-1 were made using specific rectriction sitesin the PGC-1 were made using specific rectriction sites in the PGC-1cDNA closed in pBluescript. The following restriction enzymes were usedfor these deletions: full-length Xhol (aa-1-797), Haell (aa 1-675) Ncol(aa 1-503), Xbal (aa 1-403), Kpnl (aa 1-338), and Stul (aa 1-292). Thesewere then translated into vitro with an [³⁵S]methionine-label. Onemicroliter of each in vitro translation reaction was resolved bySDS-PAGE and autoradigraphed.

[0196]FIG. 4 summarizes the input of both the full-length PGC-1 (1-797)and the 1-675 deletion which bind to the immobilized PPARγ. The bindingof PGC-1 1-503, which lacks the SR and RNA-binding domains, is modestlydecreased to 18%. A similar level of binding can be seen for PGC-1 1-403and 1-338. However, PGC-1 1-292, which still contains the LXXLL (SEQ IDNO:3) motif, completely loses the ability to interact with PPARγ. Asshown in FIG. 2A, residues 292-338 contain no distinct domains known tomediate protein-protein interaction, though it is very rich in prolineresidues.

[0197] Most of the nuclear hormone receptor coactivators identified todate interact with the C-terminal AF-2 domain, which is responsible forligand-dependent transcriptional activation. To determine if PGC-1 alsointeracts with this part of PPARγ, several deletions of PPARγ preparedas GST fusion proteins were used and combined with in vitro-translatedPGC-1. FIG. 5 shows that amino acids 181-505 of PPARγ (the originalfragment used in the yeast two-hybrid screen) interact strongly withPGC-1, pulling down 23% of the input. On the other hand, a furtherdeletion of 45 amino acids (228-505) is not able to bind to full-lengthPGC-1. Both of these PPARγ deletions were able to bind SRC-1, indicatingthat they have not lost their general ability to interact with otherproteins. These data demonstrate that PPARγ utilizes part of itsDNA-binding and hinge domains to bind PGC-1. It apparently does notinteract through the C-terminal AF-2 domain that docks othercoactivators such as SRC-1 and CBP.

Example VII Transcriptional Activity and Deletion Analysis of PGC-1

[0198] To address whether PGC-1 has its own transcriptional activationdomain or contains some activity that might unmask or augment thetranscriptional activator properties of the nuclear receptors, a numberof fusion proteins between full length or portions of PGC-1 and theDNA-binding domain (DBD) of GAL4 were prepared and assayed transcriptionthrough a GAL4 DNA binding target sequence, the UAS. Transcription wasassayed with a reporter plasmid containing five copies of the UAS linkedto CAT. More specifically, transcriptional activation assays wereperformed as follows. An expression plasmid containing full-length PGC-1was constructed by first ligating the entire 3 kb cDNA as a Small-Xholfragment into Smal-Sall sites of pSV-SPORT (GIBCO-BRL). This wasexpressed in cells with -CMX vector, along with a control fusion betweenGAL4 DBD and full-length murine SRC 1. The activity stimulated by 4.5 μgof the DBD-PGC-1 was set as 100%. The −3740/+110 bp UCP promoter wasdescribed previously (Kozak et al. (1994) Mol. Cell. Biol. 14:59-67).Rat1 IR fibroblasts were cultured in DMEM containing 10% cosmic calfserum and transfected at 80%-90% confluence by the calcium phosphatemethod. Ligands were dissolved in a vehicle containing 0.1% DMSO (9-cisretinoic acid and troglitazone) or water (8-bromocAMP). Transfectionswere performed in duplicate and repeated at least three times. CATactivity was assayed as described in Kim and Spiegelman ((1996) GenesDev. 10:1096-1107).

[0199] For GAL4 fusion constructs, full-length PGC-1 generated by PCRwas cloned in-frame into the Sall-EcoRV sites of pCMX-GAL4 plasmid.Murine full-length SRC-1 was cloned into the Smal site of RSV.GAL4.COScells were transfected in the same way as Rat1 IR fibroblasts and thereporter was the 5xUASg-CAT.

[0200] As shown in FIG. 3B, PGC-1 can activate transcription readilywhen tethered to DNA by the GAL4 DBD. For comparison, the resultsobtained by fusion of the GAL4 DBD with another coactivator of nuclearreceptors, SRC-1, is shown. Thus, PGC-1 does not absolutely requiredocking to a nuclear receptor to demonstrate transcriptional activationfunction; it is likely that its interaction with these receptors servesprimarily to bring PGC-1 to appropriate DNA sites.

[0201] To further determine the location of the transcriptionalactivation domain of PGC-1, a number of deletion mutants fused to a GAL4DNA binding domain were tested for the induction of a luciferasereporter gene as described above. The following constructs were tested:control GAL-4 alone, GAL4-PGC-1, GAL4-amino acids 1-65 of PGC-1,GAL4-amino acids 1-125 of PGC-1, GAL4-amino acids 1-170 of PGC-1,GAL4-amino acids 1-350 of PGC-1, GAL4-amino acids 1-550 of PGC-1,GAL4-amino acids 1-650 of PGC-1, GAL4-amino acids 1-650 of PGC-1, andGAL4-amino acids 170-797 of PGC-1. The results are summarized below inTable 1. TABLE 1 Transcription Activity of PGC-1-GAL-4 contructsLUCIFERASE CONSTRUCT UNITS GAL 4 Alone 4 GAL4-PGC-1 700 GAL4 1-65 4800GAL4 1-125 84,000 GAL4 1-170 36,000 GAL4 1-350 700 GAL4 1-550 4,300 GAL41-650 300 GAL4 170-797 4

[0202] As shown in Table 1, GAL4-PGC-1 constructs containing theN-terminal region of the molecule) show a higher transcriptionalactivity than the full length molecule. The construct GAL4 170-797showed no detectable transcriptional activity. These results indicatethat the transcriptional activation domain of PGC-1 is located at theN-terminal region of the molecule and in particular, at amino acids1-170 of PGC-1. The decreased in transcriptional activity observed asC-terminal amino acid residues are included (e.g., compare thetranscriptional activity of GAL4 1-125 and the full length molecule)suggests that these C-terminal residues may inhibit the transcriptionalactivity of the N-terminal domain by, e.g., masking this domain or byinteracting with other proteins which may mask or otherwise antagonizethe activity of this domain.

[0203] The above-described assay and constructs provide a useful assayfor screening compounds or agents which can modulate, e.g., stimulate orinhibit, the function of PGC-1. Particularly, preferred compounds oragent include activators of PGC-1, e.g., agents that antagonize theinhibitory effect of the C-terminal portion of the molecule. Thesecompounds or agents can be useful in modulating thermogenesis.

Example VIII Role of Protein kinase a in Modulating PGC-1 Activity

[0204] Expression of UCP genes is highly sensitive to cAMP. Analysis ofthe PGC-1 sequence revealed three consensus sites for phosphorylation byprotein kinase A (FIGS. 2A and 2B). This finding suggests a potentialrole of this kinase in regulating the activity of PGC-1, which in turnwould modify UCP gene expression. To address this possibility, sitedirected mutagenesis can be performed to ablate these phosphorylationsites. For example, amino acids 373-376 of SEQ ID NO:2 can be mutatedusing standard protocols. The transcriptional activity of the resultingmutants can be tested in, e.g., COS cells or HeLa cells, carrying areporter gene, e.g., a CAT gene, under the control of an UCP promoter.

Example IX Ectopic Expression of PGC-1 Induces Molecular Components ofAdaptive Thermogenesis

[0205] To examine directly the ability of PGC-1 to regulate the genes ofadaptive thermogenesis, retroviral vectors have been used to expressthis protein in white fat precursor cells, and 3T3-F442A preadipocyteswere then stimulated to differentiate. Briefly, the PGC-1 viralexpression vector (pBabe-PGC-1) was constructed by ligating theBamHI-Xhol fragment from pBluescript-PGC-I. plasmid into BamHI/Sallsites of pBabe-puro. Following drug selection, virally infected3T3F442A-PGC-1 and 3T3F441-vector cells lines were grown to confluencein DMEM with 10% BCS. Differentiation of these cells was initiated byculturing them in DMEM insulin. Cells were refed every 2 days with thismedium. Specific cells were grown in DMEM with 10% CCS to confluence.These cells were then treated with 1 μM dexamethasone, 0.5 mM ofmethyl-isobutyl-xanthine, 125 μM indomethacin, 17 nM insulin, and 1 nMT₃ for 48 hrs. to induce differentiation. Cells were subsequentlymaintained in DMEM containing 10% CCS, 17 μM insulin, and 1 nM T₃ andreplenished every 2 days. After these treatments, total RNA was isolatedand analyzed.

[0206] To induce UCP-1 expression, 1 μM 8-bromo-cAMP and 1 mM9-cis-retinoic acid were added to the medium, and total RNA wasextracted from the cells 6 hr later. Nothern blot analysis with a PGC-1probe revealed that PGC-1 mRNA was barely detectable in these white fatcells infected with empty vectors but was more highly expressed in cellsinfected with the viruses containing the PGC-1 cDNA. The expression ofthis mRNA in the cultured cells was approximately 6% of that seen in thebrown fat of cold exposed mice. mRNA for UCP-1, the classic marker ofbrown fat cells that is encoded in the cell nucleus, is barelydetectable in the control 3T3-F442A cells but is significantly inducedin the cells expressing PGC-1. mRNA for ATP synthesase, a keymitochondrial protein involved in oxidative phosphorylation that is alsoencoded in the nucleus, is likewise increased in the cells expressingPGC-1. The mitochondrial respiratory enzyme cytochrome c-oxidasesubunits COX II and IV are encoded in the mitochondrial and nucleargenome, respectively. Both of these mRNAs increase 2- to 3-fold in thecells ectopicaily expressing PGC-1. Expression of aP2, a white and brownfat cell gene not linked to thermogenesis, and 36B4, a ribosomal proteinare shown as a loading control. These results demonstrate that PGC-1 canstimulate the expression of several key genes of mitochondrial functionand adaptive thermogenesis, even when expressed at levels far belowthose seen in cold-exposed animals.

[0207] The ability of PGC-1 to affect the expression of mRNA for aprotein (COX-II) encoded in the mitochondrial genome suggests that PGC-1could affect the biogenesis of mitochondria per se. Changes in thecellular content of mitochondrial DNA have been used as a simplebiochemical assay for mitochondrial proliferation (Martin et al. (1995)Biochem. J. 308:749-752; Klingenspor et al. (1996) Biochem. J.316:607-613). To address this possibility, Southern blot analysis ofmitochondrial DNA was performed. 3T3-F442ASouthem blots were carried outby isolating and processing genomic DNA as described in Maniatis et al.(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press)). 3T3-F442A cellswere differentiated as described above. Total cellular DNA was isolatedand was digested with NCo I. Ten micrograms of DNA were electrophoresed,and the Southern blot was hybridized using COX-II cDNA as a probe formitrochondrial DNA.

[0208] Southern blot analysis of of mitochondrial genome DNA revealedthat cells expressing PGC-1 have twice the mitochondrial DNA contentcompared to control cells. The same blots were also probed with cDNA for36B4, a ribosomal protein encoded in the nucleus. The blot was thenstripped and hybridized with the nuclear gene 3664. These results showthat ectopic PGC-1 expression can stimulate an increase in mitochondrialDNA, indicating an increased biogenesis of mitochondria.

Example X Chronic Treatment of PGC-1 Infected Cells Increases OxygenConsumption

[0209] To determine a physiological role for PGC-1 in mediatingthermogenesis, oxygen consumption assays were performed using 3T3-F442Apreadipocytes infected with PGC-1-expressing retroviral vectors asdescribed above. The efficiency of infection is estimated to be 25%-30%of the cells. Oxygen consumption assays were performed as described inLudwik, J. et al. (1981) J. Biochemistry 256(24): 12840-12848 andHermesh, O. (1998) J. Biochemistry 273(7): 3937-3942. Treatment of thesecells with 1 μM 8-bromo-cAMP and 1 mM 9-cis-retinoic acid for 6 hoursresulted in a 100% increase in oxygen consumption by these cells (FIG.6). The increase in oxygen consumption detected in these cells is likelyto be caused by an increase in the activity and/or expression ofmitochondrial uncoupling proteins (UCPs) or similar proteins which mayfacilitate proton transport.

[0210] These experiments demonstrate that PGC-1 is capable of mediatinga thermogenic response in vivo, thus linking directly the induction ofmitochondrial DNA and gene expression to a physiological response. Thephysiological function of PGC-1 can be further characterized in tissuesknown to expressed high levels of this molecule, such as muscle. Forexample, mouse myoblast cells which can be induced to differentiate intomyotube such as C2-C12 cells, can be infected with a retrovirusexpressing PGC-1 and tested under the conditions described above.

Example XI PGC-1 is a Unique Nuclear Receptor Coactivator

[0211] The results presented herein show that PGC-1 is unusual amongknown nuclear receptor coactivators in that its expression isdramatically regulated with respect to both tissue selectivity and thephysiological state of the animal. The expression of PGC-1 in BAT butnot WAT distinguishes it from most known transcriptional components inthese tissues and its induction by cold is even more dramatic than thatobserved for UCP-1. PGC-1 is also distinct from the known coactivatorsin that it appears to use different sequence motifs for protein-proteindocking, on both sides of the receptor-coactivator pair. Nearly all ofthe known coactivators and corepressors utilize LXXLL (SEQ ID NO:3)sequences to bind at the ligand-regulated helix 12 in the carboxyterminal AF-2 domain (Heery et al. (1997) Nature 387:733-736; Torchia etal. (1997) Nature 387:677-684. In contrast, PGC-1 utilizes a domain richin proline residues to bind to a region that overlaps the DNA bindingand hinge region of PPARγ. For PPARγ , this opens the possibility thatPGC-1 is not an alternative coactivator to one or more of theligand-controlled coactivators but, rather, may bind in concert withthese proteins to give a larger macromolecular complex. On the otherhand, ligand-dependent docking is seen with some other receptors such asthe retinoic acid receptor, the estrogen receptor, and to a certaindegree, the thyroid receptor. Since PGC-1 has one LXXLL (SEQ ID NO:3)sequence, a motif shown in several contexts to be both necessary andsufficient for ligand-dependent receptor docking, it is entirelypossible that the binding of PGC-1 to those receptors will depend onthis sequence and the receptor AF-2 domains.

[0212] It is now appreciated that most of the coactivators orcorepressors that bind to receptors at AF-2 domains carry either histoneacetyltransferase or histone deacetylase activities (Pazin and Kadonaga(1997) Cell 89:325-328). These activities may be intrinsic to certaincoactivators such as CBP and SRC-1 (Bannister and Kouzarides, (1996)Nature 384:641-643; Spencer et al. (1997) Nature 389:194-198) or residein proteins that form complexes with corepressors, as illustrated by thecomplex between SMRT and mammalian histone deacetylase (Nagy et al.(1997) Cell 89:373-380; Torchia et al. (1997) Nature 387:677-684. Basedon primary sequence data, PGC-1 does not contain any motifs that wouldbe suggestive of histone acetylase or deacetylase activity. It also hasno significant sequence homologies with any of the known nuclearreceptor coactivators or corepressors. It may be noteworthy that PGC-1has paired SR and RNA-binding domains that have been identified in anumber of proteins, including several that bind to the regulatorycarbody terminal domain (CTD) of RNA polymerase II (Yuryev et al. (1996)Proc. Natl. Acad. Sci. USA 93:6975-6980. The findings presented hereincould also be explained by PGC-1 relieving a gene repression mechanism.The hinge region of at least one nuclear receptor (TR) has been shown tobe involved in binding a corepressor (N-CoR; Horlein et al. (1995)Nature 377:397404. Hence, PGC-1's action may be to derepresstranscription by interfering with corepressor binding.

Example XII Role of PGC-1 in Adaptive Thermogenesis

[0213] Adaptive thermogenesis refers to a component of energyexpenditure, which is separate from physical activity and which can beelevated in response to changing environmental conditions, most notablycold exposure and overfeeding (Himms-Hagen (1989) Proc. Soc. Exp. Biol.Med. 208:159-169). There is considerable interest in this subjectbecause of its potential roles in both the pathogenesis and therapy ofhuman obesity.

[0214] A role for PGC-1 in adaptive thermogenesis is indicated first byits connection to the key tissues and hormones implicated in thisprocess. The results shown herein suggest an especially important rolefor skeletal muscle and brown fat. PGC-1 is induced by cold exposure inboth muscle and brown fat but not in other tissues. The thermogenic andantiobesity properties of brown fat are conclusively established inrodents (Himms-Hagen (1995) Proc. Soc. exp. Biol. Med. 208:159-169), butthe role of BAT is less clear in humans due to the fact that adulthumans and other large mammals do not have well-defined brown fatdepots. The expression of UCP-1 in the white fat depots of adultssuggests that brown adipocytes may be incorporated into depots thatappear white and can be recruited upon adrenergic stimulation (Garrutiand Ricquier, (1992) Int. J. Obes. Relat. Metab. Disord. 16:383-390).

[0215] With regard to hormones, thyroid hormone and β-adrenergicagonists appear to play the most important roles in both cold anddiet-induced thermogenesis in muscle and brown fat (Himms-Hagen (1989)Proc. Soc. Exp. Biol. Med. 208:259-269; Cannon and Nedergaard (1996)Biochem. Soc. Trans. 24:407-412). β-adrenergic agonists appear to affectPGC-1 function in at least two distinct ways. First, they can inducePGC-1 expression. Second, cyclic AMP (the intracellular mediator ofβ-adrenergic receptor activity) increases the transcriptional activitymediated by PGC-1 when expression is driven ectopically, as shown inFIG. 3B. While the molecular basis of this is not known, the presence ofthree consensus phosphorylation sites for protein kinase A suggests thatthe protein may be posttranslationally activated by this pathway. Thethermogenic effects of thyroid hormone and its receptors are well known.One of the clearest effects of increasing thyroid hormone levels is thestimulation of mitochondrial respiration rates in skeletal muscle, brownfat, heart, and kidney. Abnormally low respiration rates, characteristicof a hypothyroid state, can be increased by raising thyroid hormonelevels (Pillar and Seitz (1997) Eur. J. Endocrinol. 135:231-239). Basedon the tissues where it is expressed and its ability to coactivate theTR, PGC-1 appears to be a very good candidate to mediate some of theseeffects.

[0216] Recent evidence has also suggested interesting effects of theTZDs in thermogenesis. These PPARγ ligands can increase energyexpenditure when given systematically to rodents, perhaps due toincreased formation of brown fat and an increase in Ucp-1 geneexpression. These effects have also been seen in cultured cells(Foellmi-Adams et al. (1996) Biochem. Pharmacol. 52:693-701; Tai et al.,J. Biol. Chem. 271:29909-29914 (1996). The ability of PGC-1 tocoactivate the function of PPARγ on the UCP-1 promoter, and presumablyother promoters in thermogenic pathways, may provide some explanationfor these effects.

[0217] In addition to these associations described above, ectopicexpression experiments presented here show more directly that PGC-1 canregulate components of thermogenesis. At a cellular and molecular level,adaptive thermogenesis consists of at least three separable processes:the biogenesis of mitochondria, the expression of the mitochondrialenzymes of the respiratory chain, and the expression of specificuncoupling proteins. There are now three known members of the UCP genefamily; UCP-1, expressed exclusively in brown fat; Ucp-2, expressedwidely, and Ucp-3, expressed primarily in skeletal muscle and brown fat.Depending on the length of time and severity of a given physiologicalchallenge, one or more of these aspects of thermogenesis may be affectedin muscle, BAT, or other tissues.

[0218] The retroviral expression of PGC-1 described herein have usedwhite fat cells. This cell type was chosen because it has littleendogenous PGC-1 expression and is known to have relatively low numbersof mitochondria and little expression of UCP-1 or UCP-3. Although wewere only able to get a relatively low level of PGC-1 mRNA expression(6% of that seen in cold-induced BAT), it is clear that severalmolecular components of the adaptive thermogenesis system are altered.First, expression of the Ucp-1 gene is turned on from the almostundetectable level that is characteristic of these white cells. Second,several mitochondrial genes of the respiratory chain that are ordinarilyexpressed in these cells, such as ATP synthetase, Cox-II and Cox-IV, aresignificantly increased. Finally, mitochondrial content is doubled, asevidenced by the increase in mitochondrial DNA per unit of totalcellular DNA.

[0219] The mechanism by which PGC-1 may regulate mitochondrial processeslinked to adaptive thermogenesis can be as follows. For genes such asUCP-1 that are encoded in the nucleus and are responsive to PPAR, TR, orother nuclear receptors, PGC-1 could act directly as a coactivator toincrease transcription rates. For genes that are encoded in themitochondrial genome (such as Cox-II), PGC-1 could be acting directly orindirectly. Certain genes within the mitochondria have been shown tohave functional thyroid response elements (TREs; Pillar and Seitz (1997)Eur. J. Endocrinol. 135:231-239). While PGC-1 is observed mainly in thenucleus, a small percentage of the TR and PGC-1 are transported into thenitochondria and function directly at these sites. Similarly, withregard to mitochondrial DNA replication, the D loop of the mitochondrialgenome is a site of heavy strand replication and contains a TRE-DR2sequence (Wrutniak et al., (1995) J. Biol. Chem. 270:16347-16354),suggesting that the TR and PGC-1 could act here directly. On the otherhand, PGC-1 and nuclear receptors could regulate the expression of othernuclear factors, such as NRF or mitochondrial factor A, that have beenshown to function in the mitochondria to stimulate gene transcriptionand/or DNA replication (Pillar and Seitz, (1997) Eur. J. Endocrinol.135:231-239).

[0220] The contents of all cited references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application(including the Background Section) are hereby expressly incorporated byreference. The entire contents of Appendix A (including the Figuresdepicted therein) entitled “A Cold-Inducible Coactivator of NuclearReceptors Linked to Adaptive Thermogenesis” is also incorporated byreference.

[0221] Equivalents

[0222] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 6 1 3066 DNA Mus musculus CDS (92)..(2482) 1 aattcggcac gaggttgcctgcatgagtgt gtgctgtgtg tcagagtgga ttggagttga 60 aaaagcttga ctggcgtcattcgggagctg g atg gct tgg gac atg tgc agc 112 Met Ala Trp Asp Met Cys Ser1 5 caa gac tct gta tgg agt gac ata gag tgt gct gct ctg gtt ggt gag 160Gln Asp Ser Val Trp Ser Asp Ile Glu Cys Ala Ala Leu Val Gly Glu 10 15 20gac cag cct ctt tgc cca gat ctt cct gaa ctt gac ctt tct gaa ctt 208 AspGln Pro Leu Cys Pro Asp Leu Pro Glu Leu Asp Leu Ser Glu Leu 25 30 35 gatgtg aat gac ttg gat aca gac agc ttt ctg ggt gga ttg aag tgg 256 Asp ValAsn Asp Leu Asp Thr Asp Ser Phe Leu Gly Gly Leu Lys Trp 40 45 50 55 tgtagc gac caa tcg gaa atc ata tcc aac cag tac aac aat gag cct 304 Cys SerAsp Gln Ser Glu Ile Ile Ser Asn Gln Tyr Asn Asn Glu Pro 60 65 70 gcg aacata ttt gag aag ata gat gaa gag aat gag gca aac ttg cta 352 Ala Asn IlePhe Glu Lys Ile Asp Glu Glu Asn Glu Ala Asn Leu Leu 75 80 85 gcg gtc ctcaca gag aca ctg gac agt ctc ccc gtg gat gaa gac gga 400 Ala Val Leu ThrGlu Thr Leu Asp Ser Leu Pro Val Asp Glu Asp Gly 90 95 100 ttg ccc tcattt gat gca ctg aca gat gga gcc gtg acc act gac aac 448 Leu Pro Ser PheAsp Ala Leu Thr Asp Gly Ala Val Thr Thr Asp Asn 105 110 115 gag gcc agtcct tcc tcc atg cct gac ggc acc cct ccc cct cag gag 496 Glu Ala Ser ProSer Ser Met Pro Asp Gly Thr Pro Pro Pro Gln Glu 120 125 130 135 gca gaagag ccg tct cta ctt aag aag ctc tta ctg gca cca gcc aac 544 Ala Glu GluPro Ser Leu Leu Lys Lys Leu Leu Leu Ala Pro Ala Asn 140 145 150 act cagctc agc tac aat gaa tgc agc ggt ctt agc act cag aac cat 592 Thr Gln LeuSer Tyr Asn Glu Cys Ser Gly Leu Ser Thr Gln Asn His 155 160 165 gca gcaaac cac acc cac agg atc aga aca aac cct gcc att gtt aag 640 Ala Ala AsnHis Thr His Arg Ile Arg Thr Asn Pro Ala Ile Val Lys 170 175 180 acc gagaat tca tgg agc aat aaa gcg aag agc att tgt caa cag caa 688 Thr Glu AsnSer Trp Ser Asn Lys Ala Lys Ser Ile Cys Gln Gln Gln 185 190 195 aag ccacaa aga cgt ccc tgc tca gag ctt ctc aag tat ctg acc aca 736 Lys Pro GlnArg Arg Pro Cys Ser Glu Leu Leu Lys Tyr Leu Thr Thr 200 205 210 215 aacgat gac cct cct cac acc aaa ccc aca gaa aac agg aac agc agc 784 Asn AspAsp Pro Pro His Thr Lys Pro Thr Glu Asn Arg Asn Ser Ser 220 225 230 agagac aaa tgt gct tcc aaa aag aag tcc cat aca caa ccg cag tcg 832 Arg AspLys Cys Ala Ser Lys Lys Lys Ser His Thr Gln Pro Gln Ser 235 240 245 caacat gct caa gcc aaa cca aca act tta tct ctt cct ctg acc cca 880 Gln HisAla Gln Ala Lys Pro Thr Thr Leu Ser Leu Pro Leu Thr Pro 250 255 260 gagtca cca aat gac ccc aag ggt tcc cca ttt gag aac aag act att 928 Glu SerPro Asn Asp Pro Lys Gly Ser Pro Phe Glu Asn Lys Thr Ile 265 270 275 gagcga acc tta agt gtg gaa ctc tct gga act gca ggc cta act cct 976 Glu ArgThr Leu Ser Val Glu Leu Ser Gly Thr Ala Gly Leu Thr Pro 280 285 290 295ccc aca act cct cct cat aaa gcc aac caa gat aac cct ttc aag gct 1024 ProThr Thr Pro Pro His Lys Ala Asn Gln Asp Asn Pro Phe Lys Ala 300 305 310tcg cca aag ctg aag ccc tct tgc aag acc gtg gtg cca ccg cca acc 1072 SerPro Lys Leu Lys Pro Ser Cys Lys Thr Val Val Pro Pro Pro Thr 315 320 325aag agg gcc cgg tac agt gag tgt tct ggt acc caa ggc agc cac tcc 1120 LysArg Ala Arg Tyr Ser Glu Cys Ser Gly Thr Gln Gly Ser His Ser 330 335 340acc aag aaa ggg ccc gag caa tct gag ttg tac gca caa ctc agc aag 1168 ThrLys Lys Gly Pro Glu Gln Ser Glu Leu Tyr Ala Gln Leu Ser Lys 345 350 355tcc tca ggg ctc agc cga gga cac gag gaa agg aag act aaa cgg ccc 1216 SerSer Gly Leu Ser Arg Gly His Glu Glu Arg Lys Thr Lys Arg Pro 360 365 370375 agt ctc cgg ctg ttt ggt gac cat gac tac tgt cag tca ctc aat tcc 1264Ser Leu Arg Leu Phe Gly Asp His Asp Tyr Cys Gln Ser Leu Asn Ser 380 385390 aaa acg gat ata ctc att aac ata tca cag gag ctc caa gac tct aga 1312Lys Thr Asp Ile Leu Ile Asn Ile Ser Gln Glu Leu Gln Asp Ser Arg 395 400405 caa cta gac ttc aaa gat gcc tcc tgt gac tgg cag ggg cac atc tgt 1360Gln Leu Asp Phe Lys Asp Ala Ser Cys Asp Trp Gln Gly His Ile Cys 410 415420 tct tcc aca gat tca ggc cag tgc tac ctg aga gag act ttg gag gcc 1408Ser Ser Thr Asp Ser Gly Gln Cys Tyr Leu Arg Glu Thr Leu Glu Ala 425 430435 agc aag cag gtc tct cct tgc agc acc aga aaa cag ctc caa gac cag 1456Ser Lys Gln Val Ser Pro Cys Ser Thr Arg Lys Gln Leu Gln Asp Gln 440 445450 455 gaa atc cga gcg gag ctg aac aag cac ttc ggt cat ccc tgt caa gct1504 Glu Ile Arg Ala Glu Leu Asn Lys His Phe Gly His Pro Cys Gln Ala 460465 470 gtg ttt gac gac aaa tca gac aag acc agt gaa cta agg gat ggc gac1552 Val Phe Asp Asp Lys Ser Asp Lys Thr Ser Glu Leu Arg Asp Gly Asp 475480 485 ttc agt aat gaa caa ttc tcc aaa cta cct gtg ttt ata aat tca gga1600 Phe Ser Asn Glu Gln Phe Ser Lys Leu Pro Val Phe Ile Asn Ser Gly 490495 500 cta gcc atg gat ggc cta ttt gat gac agt gaa gat gaa agt gat aaa1648 Leu Ala Met Asp Gly Leu Phe Asp Asp Ser Glu Asp Glu Ser Asp Lys 505510 515 ctg agc tac cct tgg gat ggc acg cag ccc tat tca ttg ttc gat gtg1696 Leu Ser Tyr Pro Trp Asp Gly Thr Gln Pro Tyr Ser Leu Phe Asp Val 520525 530 535 tcg cct tct tgc tct tcc ttt aac tct ccg tgt cga gac tca gtgtca 1744 Ser Pro Ser Cys Ser Ser Phe Asn Ser Pro Cys Arg Asp Ser Val Ser540 545 550 cca ccg aaa tcc tta ttt tct caa aga ccc caa agg atg cgc tctcgt 1792 Pro Pro Lys Ser Leu Phe Ser Gln Arg Pro Gln Arg Met Arg Ser Arg555 560 565 tca aga tcc ttt tct cga cac agg tcg tgt tcc cga tca cca tattcc 1840 Ser Arg Ser Phe Ser Arg His Arg Ser Cys Ser Arg Ser Pro Tyr Ser570 575 580 agg tca aga tca agg tcc cca ggc agt aga tcc tct tca aga tcctgt 1888 Arg Ser Arg Ser Arg Ser Pro Gly Ser Arg Ser Ser Ser Arg Ser Cys585 590 595 tac tac tat gaa tca agc cac tac aga cac cgc aca cac cgc aattct 1936 Tyr Tyr Tyr Glu Ser Ser His Tyr Arg His Arg Thr His Arg Asn Ser600 605 610 615 ccc ttg tat gtg aga tca cgt tca agg tca ccc tac agc cgtagg ccc 1984 Pro Leu Tyr Val Arg Ser Arg Ser Arg Ser Pro Tyr Ser Arg ArgPro 620 625 630 agg tac gac agc tat gaa gcc tat gag cac gaa agg ctc aagagg gat 2032 Arg Tyr Asp Ser Tyr Glu Ala Tyr Glu His Glu Arg Leu Lys ArgAsp 635 640 645 gaa tac cgc aaa gag cac gag aag cgg gag tct gaa agg gccaaa cag 2080 Glu Tyr Arg Lys Glu His Glu Lys Arg Glu Ser Glu Arg Ala LysGln 650 655 660 aga gag agg cag aag cag aaa gca att gaa gag cgc cgt gtgatt tac 2128 Arg Glu Arg Gln Lys Gln Lys Ala Ile Glu Glu Arg Arg Val IleTyr 665 670 675 gtt ggt aaa atc aga cct gac aca acg cgg aca gaa ttg agagac cgc 2176 Val Gly Lys Ile Arg Pro Asp Thr Thr Arg Thr Glu Leu Arg AspArg 680 685 690 695 ttt gaa gtt ttt ggt gaa att gag gaa tgc acc gta aatctg cgg gat 2224 Phe Glu Val Phe Gly Glu Ile Glu Glu Cys Thr Val Asn LeuArg Asp 700 705 710 gat gga gac agc tat ggt ttc atc acc tac cgt tac acctgt gac gct 2272 Asp Gly Asp Ser Tyr Gly Phe Ile Thr Tyr Arg Tyr Thr CysAsp Ala 715 720 725 ttc gct gct ctt gag aat gga tat act tta cgc agg tcgaac gaa act 2320 Phe Ala Ala Leu Glu Asn Gly Tyr Thr Leu Arg Arg Ser AsnGlu Thr 730 735 740 gac ttc gag ctg tac ttt tgt gga cgg aag caa ttt ttcaag tct aac 2368 Asp Phe Glu Leu Tyr Phe Cys Gly Arg Lys Gln Phe Phe LysSer Asn 745 750 755 tat gca gac cta gat acc aac tca gac gat ttt gac cctgct tcc acc 2416 Tyr Ala Asp Leu Asp Thr Asn Ser Asp Asp Phe Asp Pro AlaSer Thr 760 765 770 775 aag agc aag tat gac tct ctg gat ttt gat agt ttactg aag gaa gct 2464 Lys Ser Lys Tyr Asp Ser Leu Asp Phe Asp Ser Leu LeuLys Glu Ala 780 785 790 cag aga agc ttg cgc agg taacgtgttc ccaggctgaggaatgacaga 2512 Gln Arg Ser Leu Arg Arg 795 gagatggtca atacctcatgggacagcgtg tcctttccca agactcttgc aagtcatact 2572 taggaatttc tcctactttacactctctgt acaaaaataa aacaaaacaa aacaacaata 2632 acaacaacaa caacaacaataacaacaaca accataccag aacaagaaca acggtttaca 2692 tgaacacagc tgctgaagaggcaagagaca gaatgataat ccagtaagca cacgtttatt 2752 cacgggtgtc agctttgctttccctggagg ctcttggtga cagtgtgtgt gcgtgtgtgt 2812 gtgtgggtgt gcgtgtgtgtatgtgtgtgt gtgtacttgt ttggaaagta catatgtaca 2872 catgtgagga cttgggggcacctgaacaga acgaacaagg gcgacccctt caaatggcag 2932 catttccatg aagacacacttaaaacctac aacttcaaaa tgttcgtatt ctatacaaaa 2992 ggaaaataaa taaatataaaaaaaaaaaaa aaaaaactcg agagatctat gaatcgtaga 3052 tactgaaaaa cccc 3066 2797 PRT Mus musculus 2 Met Ala Trp Asp Met Cys Ser Gln Asp Ser Val TrpSer Asp Ile Glu 1 5 10 15 Cys Ala Ala Leu Val Gly Glu Asp Gln Pro LeuCys Pro Asp Leu Pro 20 25 30 Glu Leu Asp Leu Ser Glu Leu Asp Val Asn AspLeu Asp Thr Asp Ser 35 40 45 Phe Leu Gly Gly Leu Lys Trp Cys Ser Asp GlnSer Glu Ile Ile Ser 50 55 60 Asn Gln Tyr Asn Asn Glu Pro Ala Asn Ile PheGlu Lys Ile Asp Glu 65 70 75 80 Glu Asn Glu Ala Asn Leu Leu Ala Val LeuThr Glu Thr Leu Asp Ser 85 90 95 Leu Pro Val Asp Glu Asp Gly Leu Pro SerPhe Asp Ala Leu Thr Asp 100 105 110 Gly Ala Val Thr Thr Asp Asn Glu AlaSer Pro Ser Ser Met Pro Asp 115 120 125 Gly Thr Pro Pro Pro Gln Glu AlaGlu Glu Pro Ser Leu Leu Lys Lys 130 135 140 Leu Leu Leu Ala Pro Ala AsnThr Gln Leu Ser Tyr Asn Glu Cys Ser 145 150 155 160 Gly Leu Ser Thr GlnAsn His Ala Ala Asn His Thr His Arg Ile Arg 165 170 175 Thr Asn Pro AlaIle Val Lys Thr Glu Asn Ser Trp Ser Asn Lys Ala 180 185 190 Lys Ser IleCys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser Glu 195 200 205 Leu LeuLys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr Lys Pro 210 215 220 ThrGlu Asn Arg Asn Ser Ser Arg Asp Lys Cys Ala Ser Lys Lys Lys 225 230 235240 Ser His Thr Gln Pro Gln Ser Gln His Ala Gln Ala Lys Pro Thr Thr 245250 255 Leu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro Lys Gly Ser260 265 270 Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser Val Glu LeuSer 275 280 285 Gly Thr Ala Gly Leu Thr Pro Pro Thr Thr Pro Pro His LysAla Asn 290 295 300 Gln Asp Asn Pro Phe Lys Ala Ser Pro Lys Leu Lys ProSer Cys Lys 305 310 315 320 Thr Val Val Pro Pro Pro Thr Lys Arg Ala ArgTyr Ser Glu Cys Ser 325 330 335 Gly Thr Gln Gly Ser His Ser Thr Lys LysGly Pro Glu Gln Ser Glu 340 345 350 Leu Tyr Ala Gln Leu Ser Lys Ser SerGly Leu Ser Arg Gly His Glu 355 360 365 Glu Arg Lys Thr Lys Arg Pro SerLeu Arg Leu Phe Gly Asp His Asp 370 375 380 Tyr Cys Gln Ser Leu Asn SerLys Thr Asp Ile Leu Ile Asn Ile Ser 385 390 395 400 Gln Glu Leu Gln AspSer Arg Gln Leu Asp Phe Lys Asp Ala Ser Cys 405 410 415 Asp Trp Gln GlyHis Ile Cys Ser Ser Thr Asp Ser Gly Gln Cys Tyr 420 425 430 Leu Arg GluThr Leu Glu Ala Ser Lys Gln Val Ser Pro Cys Ser Thr 435 440 445 Arg LysGln Leu Gln Asp Gln Glu Ile Arg Ala Glu Leu Asn Lys His 450 455 460 PheGly His Pro Cys Gln Ala Val Phe Asp Asp Lys Ser Asp Lys Thr 465 470 475480 Ser Glu Leu Arg Asp Gly Asp Phe Ser Asn Glu Gln Phe Ser Lys Leu 485490 495 Pro Val Phe Ile Asn Ser Gly Leu Ala Met Asp Gly Leu Phe Asp Asp500 505 510 Ser Glu Asp Glu Ser Asp Lys Leu Ser Tyr Pro Trp Asp Gly ThrGln 515 520 525 Pro Tyr Ser Leu Phe Asp Val Ser Pro Ser Cys Ser Ser PheAsn Ser 530 535 540 Pro Cys Arg Asp Ser Val Ser Pro Pro Lys Ser Leu PheSer Gln Arg 545 550 555 560 Pro Gln Arg Met Arg Ser Arg Ser Arg Ser PheSer Arg His Arg Ser 565 570 575 Cys Ser Arg Ser Pro Tyr Ser Arg Ser ArgSer Arg Ser Pro Gly Ser 580 585 590 Arg Ser Ser Ser Arg Ser Cys Tyr TyrTyr Glu Ser Ser His Tyr Arg 595 600 605 His Arg Thr His Arg Asn Ser ProLeu Tyr Val Arg Ser Arg Ser Arg 610 615 620 Ser Pro Tyr Ser Arg Arg ProArg Tyr Asp Ser Tyr Glu Ala Tyr Glu 625 630 635 640 His Glu Arg Leu LysArg Asp Glu Tyr Arg Lys Glu His Glu Lys Arg 645 650 655 Glu Ser Glu ArgAla Lys Gln Arg Glu Arg Gln Lys Gln Lys Ala Ile 660 665 670 Glu Glu ArgArg Val Ile Tyr Val Gly Lys Ile Arg Pro Asp Thr Thr 675 680 685 Arg ThrGlu Leu Arg Asp Arg Phe Glu Val Phe Gly Glu Ile Glu Glu 690 695 700 CysThr Val Asn Leu Arg Asp Asp Gly Asp Ser Tyr Gly Phe Ile Thr 705 710 715720 Tyr Arg Tyr Thr Cys Asp Ala Phe Ala Ala Leu Glu Asn Gly Tyr Thr 725730 735 Leu Arg Arg Ser Asn Glu Thr Asp Phe Glu Leu Tyr Phe Cys Gly Arg740 745 750 Lys Gln Phe Phe Lys Ser Asn Tyr Ala Asp Leu Asp Thr Asn SerAsp 755 760 765 Asp Phe Asp Pro Ala Ser Thr Lys Ser Lys Tyr Asp Ser LeuAsp Phe 770 775 780 Asp Ser Leu Leu Lys Glu Ala Gln Arg Ser Leu Arg Arg785 790 795 3 5 PRT Mus musculus Xaas at postions 2 and 3 may be anyamino acid 3 Leu Xaa Xaa Leu Leu 1 5 4 3023 DNA Homo sapiens CDS(89)..(2482) 4 caggtggctg gttgcctgca tgagtgtgtg ctctgtgtca ctgtggattggagttgaaaa 60 agcttgactg gcgtcattca ggagctgg atg gcg tgg gac atg tgc aaccag 112 Met Ala Trp Asp Met Cys Asn Gln 1 5 gac tct gag tct gta tgg agtgac atc gag tgt gct gct ctg gtt ggt 160 Asp Ser Glu Ser Val Trp Ser AspIle Glu Cys Ala Ala Leu Val Gly 10 15 20 gaa gac cag cct ctt tgc cca gatctt cct gaa ctt gat ctt tct gaa 208 Glu Asp Gln Pro Leu Cys Pro Asp LeuPro Glu Leu Asp Leu Ser Glu 25 30 35 40 cta gat gtg aac gac ttg gat acagac agc ttt ctg ggt gga ctc aag 256 Leu Asp Val Asn Asp Leu Asp Thr AspSer Phe Leu Gly Gly Leu Lys 45 50 55 tgg tgc agt gac caa tca gaa ata atatcc aat cag tac aac aat gag 304 Trp Cys Ser Asp Gln Ser Glu Ile Ile SerAsn Gln Tyr Asn Asn Glu 60 65 70 cct tca aac ata ttt gag aag ata gat gaagag aat gag gca aac ttg 352 Pro Ser Asn Ile Phe Glu Lys Ile Asp Glu GluAsn Glu Ala Asn Leu 75 80 85 cta gca gtc ctc aca gag aca cta gac agt ctccct gtg gat gaa gac 400 Leu Ala Val Leu Thr Glu Thr Leu Asp Ser Leu ProVal Asp Glu Asp 90 95 100 gga ttg ccc tca ttt gat gcg ctg aca gat ggagac gtg acc act gac 448 Gly Leu Pro Ser Phe Asp Ala Leu Thr Asp Gly AspVal Thr Thr Asp 105 110 115 120 aat gag gct agt cct tcc tcc atg cct gacggc acc cct cca ccc cag 496 Asn Glu Ala Ser Pro Ser Ser Met Pro Asp GlyThr Pro Pro Pro Gln 125 130 135 gag gca gaa gag ccg tct cta ctt aag aagctc tta ctg gca cca gcc 544 Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys LeuLeu Leu Ala Pro Ala 140 145 150 aac act cag cta agt tat aat gaa tgc agtggt ctc agt acc cag aac 592 Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser GlyLeu Ser Thr Gln Asn 155 160 165 cat gca aat cac aat cac agg atc aga acaaac cct gca att gtt aag 640 His Ala Asn His Asn His Arg Ile Arg Thr AsnPro Ala Ile Val Lys 170 175 180 act gag aat tca tgg agc aat aaa gcg aagagt att tgt caa cag caa 688 Thr Glu Asn Ser Trp Ser Asn Lys Ala Lys SerIle Cys Gln Gln Gln 185 190 195 200 aag cca caa aga cgt ccc tgc tcg gagctt ctc aaa tat ctg acc aca 736 Lys Pro Gln Arg Arg Pro Cys Ser Glu LeuLeu Lys Tyr Leu Thr Thr 205 210 215 aac gat gac cct cct cac acc aaa cccaca gag aac aga aac agc agc 784 Asn Asp Asp Pro Pro His Thr Lys Pro ThrGlu Asn Arg Asn Ser Ser 220 225 230 aga gac aaa tgc acc tcc aaa aag aagtcc cac aca cag tcg cag tca 832 Arg Asp Lys Cys Thr Ser Lys Lys Lys SerHis Thr Gln Ser Gln Ser 235 240 245 caa cac tta caa gcc aaa cca aca acttta tct ctt cct ctg acc cca 880 Gln His Leu Gln Ala Lys Pro Thr Thr LeuSer Leu Pro Leu Thr Pro 250 255 260 gag tca cca aat gac ccc aag ggt tcccca ttt gag aac aag act att 928 Glu Ser Pro Asn Asp Pro Lys Gly Ser ProPhe Glu Asn Lys Thr Ile 265 270 275 280 gaa cgc acc tta agt gtg gaa ctctct gga act gca ggc cta act cca 976 Glu Arg Thr Leu Ser Val Glu Leu SerGly Thr Ala Gly Leu Thr Pro 285 290 295 ccc acc act cct cct cat aaa gccaac caa gat aac cct ttt agg gct 1024 Pro Thr Thr Pro Pro His Lys Ala AsnGln Asp Asn Pro Phe Arg Ala 300 305 310 tct cca aag ctg aag tcc tct tgcaag act gtg gtg cca cca cca tca 1072 Ser Pro Lys Leu Lys Ser Ser Cys LysThr Val Val Pro Pro Pro Ser 315 320 325 aag aag ccc agg tac agt gag tcttct ggt aca caa ggc aat aac tcc 1120 Lys Lys Pro Arg Tyr Ser Glu Ser SerGly Thr Gln Gly Asn Asn Ser 330 335 340 acc aag aaa ggg ccg gag caa tccgag ttg tat gca caa ctc agc aag 1168 Thr Lys Lys Gly Pro Glu Gln Ser GluLeu Tyr Ala Gln Leu Ser Lys 345 350 355 360 tcc tca gtc ctc act ggt ggacac gag gaa agg aag acc aag cgg ccc 1216 Ser Ser Val Leu Thr Gly Gly HisGlu Glu Arg Lys Thr Lys Arg Pro 365 370 375 agt ctg cgg ctg ttt ggt gaccat gac tat tgc cag tca att aat tcc 1264 Ser Leu Arg Leu Phe Gly Asp HisAsp Tyr Cys Gln Ser Ile Asn Ser 380 385 390 aaa acg gaa ata ctc att aatata tca cag gag ctc caa gac tct aga 1312 Lys Thr Glu Ile Leu Ile Asn IleSer Gln Glu Leu Gln Asp Ser Arg 395 400 405 caa cta gaa aat aaa gat gtctcc tct gat tgg cag ggg cag att tgt 1360 Gln Leu Glu Asn Lys Asp Val SerSer Asp Trp Gln Gly Gln Ile Cys 410 415 420 tct tcc aca gat tca gac cagtgc tac ctg aga gag act ttg gag gca 1408 Ser Ser Thr Asp Ser Asp Gln CysTyr Leu Arg Glu Thr Leu Glu Ala 425 430 435 440 agc aag cag gtc tct ccttgc agc aca aga aaa cag ctc caa gac cag 1456 Ser Lys Gln Val Ser Pro CysSer Thr Arg Lys Gln Leu Gln Asp Gln 445 450 455 gaa atc cga gcc gag ctgaac aag cac ttc ggt cat ccc agt caa gct 1504 Glu Ile Arg Ala Glu Leu AsnLys His Phe Gly His Pro Ser Gln Ala 460 465 470 gtt ttt gac gac gaa gcagac aag acc ggt gaa ctg agg gac agt gat 1552 Val Phe Asp Asp Glu Ala AspLys Thr Gly Glu Leu Arg Asp Ser Asp 475 480 485 ttc agt aat gaa caa ttctcc aaa cta cct atg ttt ata aat tca gga 1600 Phe Ser Asn Glu Gln Phe SerLys Leu Pro Met Phe Ile Asn Ser Gly 490 495 500 cta gcc atg gat ggc ctgttt gat gac agc gaa gat aaa agt gat aaa 1648 Leu Ala Met Asp Gly Leu PheAsp Asp Ser Glu Asp Lys Ser Asp Lys 505 510 515 520 ctg agc tac cct tgggat ggc acg caa tcc tat tca ttg ttc aat gtg 1696 Leu Ser Tyr Pro Trp AspGly Thr Gln Ser Tyr Ser Leu Phe Asn Val 525 530 535 tct cct tct tgt tcttct ttt aac tct cca tgt aga gat tct gtg tca 1744 Ser Pro Ser Cys Ser SerPhe Asn Ser Pro Cys Arg Asp Ser Val Ser 540 545 550 cca ccc aaa tcc ttattt tct caa aga ccc caa agg atg cgc tct cgt 1792 Pro Pro Lys Ser Leu PheSer Gln Arg Pro Gln Arg Met Arg Ser Arg 555 560 565 tca agg tcc ttt tctcga cac agg tcg tgt tcc cga tca cca tat tcc 1840 Ser Arg Ser Phe Ser ArgHis Arg Ser Cys Ser Arg Ser Pro Tyr Ser 570 575 580 agg tca aga tca aggtct cca ggc agt aga tcc tct tca aga tcc tgc 1888 Arg Ser Arg Ser Arg SerPro Gly Ser Arg Ser Ser Ser Arg Ser Cys 585 590 595 600 tat tac tat gagtca agc cac tac aga cac cgc acg cac cga aat tct 1936 Tyr Tyr Tyr Glu SerSer His Tyr Arg His Arg Thr His Arg Asn Ser 605 610 615 ccc ttg tat gtgaga tca cgt tca aga tcg ccc tac agc cgt cgg ccc 1984 Pro Leu Tyr Val ArgSer Arg Ser Arg Ser Pro Tyr Ser Arg Arg Pro 620 625 630 agg tat gac agctac gag gaa tat cag cac gag agg ctg aag agg gaa 2032 Arg Tyr Asp Ser TyrGlu Glu Tyr Gln His Glu Arg Leu Lys Arg Glu 635 640 645 gaa tat cgc agagag tat gag aag cga gag tct gag agg gcc aag caa 2080 Glu Tyr Arg Arg GluTyr Glu Lys Arg Glu Ser Glu Arg Ala Lys Gln 650 655 660 agg gag agg cagagg cag aag gca att gaa gag cgc cgt gtg att tat 2128 Arg Glu Arg Gln ArgGln Lys Ala Ile Glu Glu Arg Arg Val Ile Tyr 665 670 675 680 gtc ggt aaaatc aga cct gac aca aca cgg aca gaa ctg agg gac cgt 2176 Val Gly Lys IleArg Pro Asp Thr Thr Arg Thr Glu Leu Arg Asp Arg 685 690 695 ttt gaa gttttt ggt gaa att gag gag tgc aca gta aat ctg cgg gat 2224 Phe Glu Val PheGly Glu Ile Glu Glu Cys Thr Val Asn Leu Arg Asp 700 705 710 gat gga gacagc tat ggt ttc att acc tac cgt tat acc tgt gat gct 2272 Asp Gly Asp SerTyr Gly Phe Ile Thr Tyr Arg Tyr Thr Cys Asp Ala 715 720 725 ttt gct gctctt gaa aat gga tac act ttg cgc agg tca aac gaa act 2320 Phe Ala Ala LeuGlu Asn Gly Tyr Thr Leu Arg Arg Ser Asn Glu Thr 730 735 740 gac ttt gagctg tac ttt tgt gga cgc aag caa ttt ttc aag tct aac 2368 Asp Phe Glu LeuTyr Phe Cys Gly Arg Lys Gln Phe Phe Lys Ser Asn 745 750 755 760 tat gcagac cta gat tca aac tca gat gac ttt gac cct gct tcc acc 2416 Tyr Ala AspLeu Asp Ser Asn Ser Asp Asp Phe Asp Pro Ala Ser Thr 765 770 775 aag agcaag tat gac tct ctg gat ttt gat agt tta ctg aaa gaa gct 2464 Lys Ser LysTyr Asp Ser Leu Asp Phe Asp Ser Leu Leu Lys Glu Ala 780 785 790 cag agaagc ttg cgc agg taacatgttc cctagctgag gatgacagag 2512 Gln Arg Ser LeuArg Arg 795 ggatggcgaa tacctcatgg gacagcgcgt ccttccctaa agactattgcaagtcatact 2572 taggaatttc tcctacttta cactctctgt acaaaaacaa aacaaaacaacaacaataca 2632 acaagaacaa caacaacaat aacaacaatg gtttacatga acacagctgctgaagaggca 2692 agagacagaa tgatatccag taagcacatg tttattcatg ggtgtcagctttgcttttcc 2752 tggagtctct tggtgatgga gtgtgcgtgt gtgcatgtat gtgtgtgtgtatgtatgtgt 2812 gtggtgtgtg tgcttggttt aggggaagta tgtgtgggta catgtgaggactgggggcac 2872 ctgaccagaa tgcgcaaggg caaaccattt caaatggcag cagttccatgaagacacact 2932 taaaacctag aacttcaaaa tgttcgtatt ctattcaaaa ggaaaaatatatatatatat 2992 atatatatat aaattaaaaa aaaaaaaaaa a 3023 5 798 PRT Homosapiens 5 Met Ala Trp Asp Met Cys Asn Gln Asp Ser Glu Ser Val Trp SerAsp 1 5 10 15 Ile Glu Cys Ala Ala Leu Val Gly Glu Asp Gln Pro Leu CysPro Asp 20 25 30 Leu Pro Glu Leu Asp Leu Ser Glu Leu Asp Val Asn Asp LeuAsp Thr 35 40 45 Asp Ser Phe Leu Gly Gly Leu Lys Trp Cys Ser Asp Gln SerGlu Ile 50 55 60 Ile Ser Asn Gln Tyr Asn Asn Glu Pro Ser Asn Ile Phe GluLys Ile 65 70 75 80 Asp Glu Glu Asn Glu Ala Asn Leu Leu Ala Val Leu ThrGlu Thr Leu 85 90 95 Asp Ser Leu Pro Val Asp Glu Asp Gly Leu Pro Ser PheAsp Ala Leu 100 105 110 Thr Asp Gly Asp Val Thr Thr Asp Asn Glu Ala SerPro Ser Ser Met 115 120 125 Pro Asp Gly Thr Pro Pro Pro Gln Glu Ala GluGlu Pro Ser Leu Leu 130 135 140 Lys Lys Leu Leu Leu Ala Pro Ala Asn ThrGln Leu Ser Tyr Asn Glu 145 150 155 160 Cys Ser Gly Leu Ser Thr Gln AsnHis Ala Asn His Asn His Arg Ile 165 170 175 Arg Thr Asn Pro Ala Ile ValLys Thr Glu Asn Ser Trp Ser Asn Lys 180 185 190 Ala Lys Ser Ile Cys GlnGln Gln Lys Pro Gln Arg Arg Pro Cys Ser 195 200 205 Glu Leu Leu Lys TyrLeu Thr Thr Asn Asp Asp Pro Pro His Thr Lys 210 215 220 Pro Thr Glu AsnArg Asn Ser Ser Arg Asp Lys Cys Thr Ser Lys Lys 225 230 235 240 Lys SerHis Thr Gln Ser Gln Ser Gln His Leu Gln Ala Lys Pro Thr 245 250 255 ThrLeu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro Lys Gly 260 265 270Ser Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser Val Glu Leu 275 280285 Ser Gly Thr Ala Gly Leu Thr Pro Pro Thr Thr Pro Pro His Lys Ala 290295 300 Asn Gln Asp Asn Pro Phe Arg Ala Ser Pro Lys Leu Lys Ser Ser Cys305 310 315 320 Lys Thr Val Val Pro Pro Pro Ser Lys Lys Pro Arg Tyr SerGlu Ser 325 330 335 Ser Gly Thr Gln Gly Asn Asn Ser Thr Lys Lys Gly ProGlu Gln Ser 340 345 350 Glu Leu Tyr Ala Gln Leu Ser Lys Ser Ser Val LeuThr Gly Gly His 355 360 365 Glu Glu Arg Lys Thr Lys Arg Pro Ser Leu ArgLeu Phe Gly Asp His 370 375 380 Asp Tyr Cys Gln Ser Ile Asn Ser Lys ThrGlu Ile Leu Ile Asn Ile 385 390 395 400 Ser Gln Glu Leu Gln Asp Ser ArgGln Leu Glu Asn Lys Asp Val Ser 405 410 415 Ser Asp Trp Gln Gly Gln IleCys Ser Ser Thr Asp Ser Asp Gln Cys 420 425 430 Tyr Leu Arg Glu Thr LeuGlu Ala Ser Lys Gln Val Ser Pro Cys Ser 435 440 445 Thr Arg Lys Gln LeuGln Asp Gln Glu Ile Arg Ala Glu Leu Asn Lys 450 455 460 His Phe Gly HisPro Ser Gln Ala Val Phe Asp Asp Glu Ala Asp Lys 465 470 475 480 Thr GlyGlu Leu Arg Asp Ser Asp Phe Ser Asn Glu Gln Phe Ser Lys 485 490 495 LeuPro Met Phe Ile Asn Ser Gly Leu Ala Met Asp Gly Leu Phe Asp 500 505 510Asp Ser Glu Asp Lys Ser Asp Lys Leu Ser Tyr Pro Trp Asp Gly Thr 515 520525 Gln Ser Tyr Ser Leu Phe Asn Val Ser Pro Ser Cys Ser Ser Phe Asn 530535 540 Ser Pro Cys Arg Asp Ser Val Ser Pro Pro Lys Ser Leu Phe Ser Gln545 550 555 560 Arg Pro Gln Arg Met Arg Ser Arg Ser Arg Ser Phe Ser ArgHis Arg 565 570 575 Ser Cys Ser Arg Ser Pro Tyr Ser Arg Ser Arg Ser ArgSer Pro Gly 580 585 590 Ser Arg Ser Ser Ser Arg Ser Cys Tyr Tyr Tyr GluSer Ser His Tyr 595 600 605 Arg His Arg Thr His Arg Asn Ser Pro Leu TyrVal Arg Ser Arg Ser 610 615 620 Arg Ser Pro Tyr Ser Arg Arg Pro Arg TyrAsp Ser Tyr Glu Glu Tyr 625 630 635 640 Gln His Glu Arg Leu Lys Arg GluGlu Tyr Arg Arg Glu Tyr Glu Lys 645 650 655 Arg Glu Ser Glu Arg Ala LysGln Arg Glu Arg Gln Arg Gln Lys Ala 660 665 670 Ile Glu Glu Arg Arg ValIle Tyr Val Gly Lys Ile Arg Pro Asp Thr 675 680 685 Thr Arg Thr Glu LeuArg Asp Arg Phe Glu Val Phe Gly Glu Ile Glu 690 695 700 Glu Cys Thr ValAsn Leu Arg Asp Asp Gly Asp Ser Tyr Gly Phe Ile 705 710 715 720 Thr TyrArg Tyr Thr Cys Asp Ala Phe Ala Ala Leu Glu Asn Gly Tyr 725 730 735 ThrLeu Arg Arg Ser Asn Glu Thr Asp Phe Glu Leu Tyr Phe Cys Gly 740 745 750Arg Lys Gln Phe Phe Lys Ser Asn Tyr Ala Asp Leu Asp Ser Asn Ser 755 760765 Asp Asp Phe Asp Pro Ala Ser Thr Lys Ser Lys Tyr Asp Ser Leu Asp 770775 780 Phe Asp Ser Leu Leu Lys Glu Ala Gln Arg Ser Leu Arg Arg 785 790795 6 27 DNA Homo sapiens 6 atcttcgctg tcatcaaaca ggccatc 27

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence encoding PGC-1 or a portion thereof.
 2. An isolatednucleic acid molecule comprising a nucleotide sequence encoding aprotein or a portion thereof, wherein the protein or portion thereofcomprises an amino acid sequence which is sufficiently homologous to anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:5 such that the proteinor portion thereof maintains the ability to modulate one or more of thefollowing biological activities: UCP expression, thermogenesis inadipose cells, differentiation of adipose cells, and insulin sensitivityof adipose cells.
 3. The isolated nucleic acid molecule of claim 2,wherein the protein comprises an amino acid sequence at least about 60%homologous to the entire amino acid sequence of SEQ ID NO:2 or SEQ IDNO:5.
 4. The isolated nucleic acid molecule of claim 2, wherein theportion of the protein comprises one or more of the following domains ormotifs: a) a cAMP phosphorylation site; b) a tyrosine phosphorylationsite; c) an RNA binding motif; d) a serine-arginine rich domain; and e)an LXXLL motif.
 5. An isolated nucleic acid molecule at least 15nucleotides in length which hybridizes to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4.
 6. Anisolated nucleic acid molecule comprising the nucleotide sequence of SEQID NO:1 or SEQ ID NO:4 or a nucleotide sequence which is at least about60% homologous to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4.7. An isolated nucleic acid molecule encoding the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:5 or an amino acid sequence which is at leastabout 60% homologous to the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:5.
 8. An isolated nucleic acid molecule encoding a PGC-1 fusionprotein.
 9. An isolated nucleic acid molecule which is antisense to thenucleic acid molecule of claim
 1. 10. A vector comprising a nucleotidesequence encoding PGC-1.
 11. A host cell containing the vector of claim10.
 12. A method for producing PGC-1 comprising culturing the host cellof claim 11 in a suitable medium until PGC-1 is produced.
 13. Anisolated PGC-1 protein or a portion thereof which can modulate one ormore of the following biological activities: UCP expression,thermogenesis in adipose cells, differentiation of adipose cells, andinsulin sensitivity of adipose cells.
 14. An isolated protein or aportion thereof which comprises an amino acid sequence which issufficiently homologous to an amino acid sequence of SEQ ID NO:2 or SEQID NO:5 such that the protein or portion thereof maintains the abilityto modulate one or more of the following biological activities: UCPexpression, thermogenesis in adipose cells, differentiation of adiposecells, and insulin sensitivity of adipose cells.
 15. The isolatedprotein or portion thereof of claim 14, wherein the portion of theprotein comprises one or more of the following domains or motifs: a) acAMP phosphorylation site; b) a tyrosine phosphorylation site; c) an RNAbinding motif; d) a serine-arginine rich domain; and e) an LXXLL motif.16. An isolated protein comprising the amino acid sequence of SEQ IDNO:2 or SEQ ID NO:5 or an amino acid sequence which is at least about60% homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.17. A fusion protein comprising a PGC-1 polypeptide operatively linkedto a non-PGC-1 polypeptide.
 18. An antigenic peptide of PGC-1 comprisingat least 8 amino acid residues of the amino acid sequence shown in SEQID NO:2 or SEQ ID NO:5, the peptide comprising an epitope of PGC-1 suchthat an antibody raised against the peptide forms a specific immunecomplex with PGC-1.
 19. An antibody that specifically binds PGC-1.
 20. Amethod for detecting the presence of PGC-1 in a biological samplecomprising contacting a biological sample with an agent capable ofdetecting PGC-1 protein or mRNA.
 21. A kit for detecting the presence ofPGC-1 in a biological sample comprising a labeled or labelable agentcapable of detecting PGC-1 protein or mRNA in a biological sample; meansfor determining the amount of PGC-1 in the sample; and means forcomparing the amount of PGC-1 in the sample with a standard.
 22. Amethod for identifying a compound capable of treating a disordercharacterized by aberrant PGC-1 nucleic acid expression or PGC-1 proteinactivity comprising assaying the ability of the compound or agent tomodulate the expression of PGC-1 nucleic acid or the activity of thePGC-1 protein thereby identifying a compound capable of treating adisorder characterized by aberrant PGC-1 nucleic acid expression orPGC-1 protein activity.
 23. A method for identifying a compound whichbinds to PGC-1 protein comprising contacting the PGC-1 protein with thecompound under conditions which allow binding of the compound to thePGC-1 protein to form a complex; and detecting the formation of acomplex of the PGC-1 protein and the compound in which the ability ofthe compound to bind to the PGC-1 protein is indicated by the presenceof the compound in the complex.
 24. A method for identifying a compoundwhich inhibits the interaction of the PGC-1 protein with a targetmolecule comprising contacting, in the presence of the compound, thePGC-1 protein and the target molecule under conditions which allowbinding of the target molecule to the PGC-1 protein to form a complex;and detecting the formation of a complex of the PGC-1 protein and thetarget molecule in which the ability of the compound to inhibitinteraction between the PGC-1 protein and the target molecule isindicated by a decrease in complex formation as compared to the amountof complex formed in the absence of the compound.